Human phospholipases

ABSTRACT

The invention provides human lipid metabolism enzymes (LME) and polynucleotides which identify and encode LME. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of LME.

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences of lipidmetabolism enzymes and to the use of these sequences in the diagnosis,treatment, and prevention of cancer, neurological disorders,autoimmune/inflammatory disorders, gastrointestinal disorders, andcardiovascular disorders, and in the assessment of the effects ofexogenous compounds on the expression of nucleic acid and amino acidsequences of lipid metabolism enzymes.

BACKGROUND OF THE INVENTION

Lipids are water-insoluble, oily or greasy substances that are solublein nonpolar solvents such as chloroform or ether. Neutral fats(triacylglycerols) serve as major fuels and energy stores. Polar lipids,such as phospholipids, sphingolipids, glycolipids, and cholesterol, arekey structural components of cell membranes. (Lipid metabolism isreviewed in Stryer, L. (1995) Biochemistry, W. H. Freeman and Company,New York N.Y.; Lehninger, A. (1982) Principles of Biochemistry, WorthPublishers, Inc. New York N.Y.; and ExPASy “Biochemical Pathways” indexof Boehringer Mannheim online publication.)

Fatty acids are long-chain organic acids with a single carboxyl groupand a long non-polar hydrocarbon tail. Long-chain fatty acids areessential components of glycolipids, phospholipids, and cholesterol,which are building blocks for biological membranes, and oftriglycerides, which are biological fuel molecules. Long-chain fattyacids are also substrates for eicosanoid production, and are importantin the functional modification of certain complex carbohydrates andproteins. 16-carbon and 18-carbon fatty acids are the most common. Fattyacid synthesis occurs in the cytoplasm. In the first step,acetyl-Coenzyme A (CoA) carboxylase (ACC) synthesizes malonyl-CoA fromacetyl-CoA and bicarbonate. The enzymes which catalyze the remainingreactions are covalently linked into a single polypeptide chain,referred to as the multifunctional enzyme fatty acid synthase (FAS). FAScatalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA FAScontains acetyl transferase, malonyl transferase, β-ketoacetyl synthase,acyl carrier protein, β-ketoacyl reductase, dehydratase, enoylreductase, and thioesterase activities. The final product of the FASreaction is the 16-carbon fatty acid palmitate. Further elongation, aswell as unsaturation, of palmitate by accessory enzymes of the ERproduces the variety of long chain fatty acids required by theindividual cell. These enzymes include a NADH-cytochrome b₅ reductase,cytochrome b₅, and a desaturase.

Triacylglycerols, also known as triglycerides and neutral fats, aremajor energy stores in animals. Triacylglycerols are esters of glycerolwith three fatty acid chains. Glycerol-3-phosphate is produced fromdihydroxyacetone phosphate by the enzyme glycerol phosphatedehydrogenase or from glycerol by glycerol kinase. Fatty acid-CoA's areproduced from fatty acids by fatty acyl-CoA synthetases.Glyercol-3-phosphate is acylated with two fatty acyl-CoA's by the enzymeglycerol phosphate acyltransferase to give phosphatidate. Phosphatidatephosphatase converts phosphatidate to diacylglycerol, which issubsequently acylated to a triacylglyercol by the enzyme diglycerideacyltransferase. Phosphatidate phosphatase and diglycerideacyltransferase form a triacylglyerol synthetase complex bound to the ERmembrane.

A major class of phospholipids are the phosphoglycerides, which arecomposed of a glycerol backbone, two fatty acid chains, and aphosphorylated alcohol. Phosphoglycerides are components of cellmembranes. Principal phosphoglycerides are phosphatidyl choline,phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol,and diphosphatidyl glycerol. Many enzymes involved in phosphoglyceridesynthesis are associated with membranes (Meyers, R. A. (1995) MolecularBiology and Biotechnology, VCH Publishers Inc., New York N.Y., pp.494-501). Phosphatidate is converted to CDP-diacylglycerol by the enzymephosphatidate cytidylyltransferase (ExPASy ENZYME EC 2.7.7.41). Transferof the diacylglycerol group from CDP-diacylglycerol to serine to yieldphosphatidyl serine, or to inositol to yield phosphatidyl inositol, iscatalyzed by the enzymes CDP-diacylglycerol-serineO-phosphatidyltransferase and CDP-diacylglycerol-inositol3-phosphatidyltransferase, respectively (ExPASy ENZYME EC 2.7.8.8;ExPASy ENZYME EC 2.7.8.11). The enzyme phosphatidyl serine decarboxylasecatalyzes the conversion of phosphatidyl serine to phosphatidylethanolamine, using a pyruvate cofactor (Voelker, D. R. (1997) Biochim.Biophys. Acta 1348:236-244). Phosphatidyl choline is formed usingdiet-derived choline by the reaction of CDP-choline with1,2-diacylglycerol, catalyzed by diacylglycerolcholinephosphotransferase (ExPASy ENZYME 2.7.8.2).

Cholesterol, composed of four fused hydrocarbon rings with an alcohol atone end, moderates the fluidity of membranes in which it isincorporated. In addition, cholesterol is used in the synthesis ofsteroid hormones such as cortisol, progesterone, estrogen, andtestosterone. Bile salts derived from cholesterol facilitate thedigestion of lipids. Cholesterol in the skin forms a barrier thatprevents excess water evaporation from the body. Farnesyl andgeranylgeranyl groups, which are derived from cholesterol biosynthesisintermediates, are post-translationally added to signal transductionproteins such as Ras and protein-targeting proteins such as Rab. Thesemodifications are important for the activities of these proteins(Guyton, A. C. (1991) Textbook of Medical Physiology, W. B. SaundersCompany, Philadelphia Pa., pp. 760-763; Stryer, supra, pp. 279-280,691-702, 934). Mammals obtain cholesterol derived from both de novobiosynthesis and the diet.

Sphingolipids are an important class of membrane lipids that containsphingosine, a long chain amino alcohol. They are composed of onelong-chain fatty acid, one polar head alcohol, and sphingosine orsphingosine derivatives. The three classes of sphingolipids aresphingomyelins, cerebrosides, and gangliosides. Sphingomyelins, whichcontain phosphocholine or phosphoethanolamine as their head group, areabundant in the myelin sheath surrounding nerve cells.Galactocerebrosides, which contain a glucose or galactose head group,are characteristic of the brain. Other cerebrosides are found innon-neural tissues. Gangliosides, whose head groups contain multiplesugar units, are abundant in the brain, but are also found in non-neuraltissues.

Eicosanoids, including prostaglandins, prostacyclin, thromboxanes, andleukotrienes, are 20-carbon molecules derived from fatty acids.Eicosanoids are signaling molecules which have roles in pain, fever, andinflammation. The precursor of all eicosanoids is arachidonate, which isgenerated from phospholipids by phospholipase A₂ and fromdiacylglycerols by diacylglycerol lipase. Leukotrienes are produced fromarachidonate by the action of lipoxygenases.

Within cells, fatty acids are transported by cytoplasmic fatty acidbinding proteins (Online Mendelian Inheritance in Man (OMIM) *134650Fatty Acid-Binding Protein 1, Liver; FABP1). Diazepam binding inhibitor(DBI), also known as endozepine and acyl CoA-binding proteins is anendogenous γ-aminobutyric acid (GABA) receptor ligand which is thoughtto down-regulate the effects of GABA. DBI binds medium- and long-chainacyl-CoA esters with very high affinity and may function as anintracellular carrier of acyl-CoA esters (OMIM *25950 Diazepam BindingInhibitor; DBI; PROSITE PDOC00686 Acyl-CoA-binding protein signature).

Fat stored in liver and adipose triglycerides may be released byhydrolysis and transported in the blood. Free fatty acids aretransported in the blood by albumin. Triacylglycerols and cholesterolesters in the blood are transported in lipoprotein particles. Theparticles consist of a core of hydrophobic lipids surrounded by a shellof polar lipids and apolipoproteins. The protein components serve in thesolubilization of hydrophobic lipids and also contain cell-targetingsignals. Lipoproteins include chylomicrons, chylomicron remnants,very-low-density lipoproteins (VLDL), intermediate-density lipoproteins(IDL), low-density lipoproteins (LDL), and high-density lipoproteins(HDL). There is a strong inverse correlation between the levels ofplasma HDL and risk of premature coronary heart disease.

Mitochondrial and peroxisomal beta-oxidation enzymes degrade saturatedand unsaturated fatty acids by sequential removal of two-carbon unitsfrom CoA-activated fatty acids. The main beta-oxidation pathway degradesboth saturated and unsaturated fatty acids while the auxiliary pathwayperforms additional steps reed for the degradation of unsaturated fattyacids. The pathways of mitochondrial and peroxisomal beta-oxidation usesimilar enzymes, but have different substrate specificities andfunctions. Mitochondria oxidize short-, medium-, and long-chain fattyacids to produce energy for cells. Mitochondrial beta-oxidation is amajor energy source for cardiac and skeletal muscle. In liver, itprovides ketone bodies to the peripheral circulation when glucose levelsare low as in starvation, endurance exercise, and diabetes (Eaton, S. etal. (1996) Biochem. J. 320:345-357). Peroxisomes oxidize medium-, long-,and very-long-chain fatty acids, dicarboxylic fatty acids, branchedfatty acids, prostaglandins, xenobiotics, and bile acid intermediates.The chief roles of peroxisomal beta-oxidation are to shorten toxiclipophilic carboxylic acids to facilitate their excretion and to shortenvery-long-chain fatty acids prior to mitochondrial beta-oxidation(Mannaerts, G. P. and P. P. Van Veldhoven (1993) Biochimie 75:147-158).Enzymes involved in beta-oxidation include acyl CoA synthetase,carnitine acyltransferase, acyl CoA dehydrogenases, enoyl CoAhydratases, L-3-hydroxyacyl CoA dehydrogenase, β-ketothiolase,2,4-dienoyl CoA reductase, and isomerase.

Three classes of lipid metabolism enzymes are discussed in furtherdetail. The three classes are lipases, phospholipases and lipoxygenases.

Lipases

Triglycerides are hydrolyzed to fatty acids and glycerol by lipases.Adipocytes contain lipases that break down stored triacylglycerols,releasing fatty adds for export to other tissues where they are requiredas fuel. Lipases are widely distributed in animals, plants, andprokaryotes. Triglyceride lipases (ExPASy ENZYME EC 3.1.1.3), also knownas triacylglycerol lipases and tributyrases, hydrolyze the ester bond oftriglycerides. In higher vertebrates there are at least threetissue-specific isozymes including gastric, hepatic, and pancreaticlipases. These three types of lipases are structurally closely relatedto each other as well as to lipoprotein lipase. The most conservedregion in gastric, hepatic, and pancreatic lipases is centered around aserine residue which is also present in lipases of prokaryotic origin.Mutation in the serine residue renders the enzymes inactive. Gastric,hepatic, and pancreatic lipases hydrolyze lipoprotein triglycerides andphospholipids. Gastric lipases in the intestine aid in the digestion andabsorption of dietary fats. Hepatic lipases are bound to and act at theendothelial surfaces of hepatic tissues. Hepatic lipases also play amajor role in the regulation of plasma lipids. Pancreatic lipaserequires a small protein cofactor, colipase, for efficient dietary lipidhydrolysis. Colipase binds to the C-terminal, non-catalytic domain oflipase, thereby stabilizing an active conformation and considerablyincreasing the overall hydrophobic binding site. Deficiencies of theseenzymes have been identified in man, and all are associated withpathologic levels of circulating lipoprotein particles (Gargouri, Y. etal. (1989) Biochim. Biophys. Acta 1006:255-271; Connelly, P. W. (1999)Clin. Chim. Acta 286:243-255; van Tilbeurgh, H. et al. (1999) BiochimBiophys Acta 1441:173-184).

Lipoprotein lipases (ExPASy ENZYME EC 3.1.1.34), also known as clearingfactor lipases, diglyceride lipases, or diacylglycerol lipases,hydrolyze triglycerides and phospholipids present in circulating plasmalipoproteins, including chylomicrons, very low and intermediate densitylipoproteins, and high-density lipoproteins (HDL). Together withpancreatic and hepatic lipases, lipoprotein lipases (LPL) share a highdegree of primary sequence homology. Both lipoprotein lipases andhepatic lipases are anchored to the capillary endothelium viaglycosaminoglycans and can be released by intravenous administration ofheparin. LPLs are primarily synthesized by adipocytes, muscle cells, andmacrophages. Catalytic activities of LPLs are activated byapolipoprotein C-II and are inhibited by high ionic strength conditionssuch as 1 M NaCl. LPL deficiencies in humans contribute to metabolicdiseases such as hypertriglyceridemia, HDL2 deficiency, and obesity(Jackson, R. L. (1983) in The Enzymes (Boyer, P. D., ed.) Vol. XVI, pp.141-186, Academic Press, New York N.Y.; Eckel, R. H. (1989) New Engl. J.Med. 320:1060-1068).

Phospholipases

Phospholipases, a group of enzymes that catalyze the hydrolysis ofmembrane phospholipids, are classified according to the bond cleaved ina phospholipid. They are classified into PLA1, PLA2, PLB, PLC, and PLDfamilies. Phospholipases are involved in many inflammatory reactions bymaking arachidonate available for eicosanoid biosynthesis. Morespecifically, arachidonic acid is processed into bioactive lipidmediators of inflammation such as lyso-platelet-activating factor andeicosanoids. The synthesis of arachidonic acid from membranephospholipids is the rate-limiting step in the biosynthesis of the fourmajor classes of eicosanoids (prostaglandins, prostacyclins,thromboxanes and leukotrienes) which are involved in pain, fever, andinflammation (Kaiser, E. et al. (1990) Clin. Biochem. 23:349-370).Furthermore, leukotriene-B4 is known to function in a feedback loopwhich further increases PLA2 activity (Wijkander, J. et al. (1995) J.Biol. Chem. 270:26543-26549).

The secretory phospholipase A₂ (PLA2) superfamily comprises a number ofheterogeneous enzymes whose common feature is to hydrolyze the sn-2fatty acid acyl ester bond of phosphoglycerides. Hydrolysis of theglycerophospholipids releases free fatty acids and lysophospholipids.PLA2 activity generates precursors for the biosynthesis of biologicallyactive lipids, hydroxy fatty acids, and platelet-activating factor.PLA2s were first described as components of snake venoms, and were latercharacterized in numerous species, PLA2s have traditionally beenclassified into several major groups and subgroups based on their aminoacid sequences, divalent cation requirements, and location of disulfidebonds. The PLA2s of Groups I, II, and III consist of low molecularweight, secreted, Ca²⁺-dependent proteins. Group IV PLA2s are primarily85-kDa, Ca²⁺-dependent cytosolic phospholipases. Finally, a number ofCa²⁺-independent PLA2s have been described, which comprise Group V(Davidson, F. F. and E. A. Dennis (1990) J. Mol. Evol. 31:228-238; andDennis, E. F. (1994) J. Biol Chem. 269:13057-13060).

The first PLA2s to be extensively characterized were the Group I, II,and III PLA2s found in snake and bee venoms. These venom PLA2s sharemany features with mammalian PLA2s including a common catalyticmechanism, the same Ca²⁺ requirement, and conserved primary and tertiarystructures. In addition to their role in the digestion of prey, thevenom PLA2s display neurotoxic, myotoxic, anticoagulant, andproinflammatory effects in mammalian tissues. This diversity ofpathophysiological effects is due to the presence of specific, highaffinity receptors for these enzymes on various cells and tissues(Lambeau, G. et al. (1995) J. Biol. Chem. 270:5534-5540).

PLA2s from Groups I, IIA, IIC, and V have been described in mammalianand avian cells, and were originally characterized by tissuedistribution, although the distinction is no longer absolute. Thus,Group I PLA2s were found in the pancreas, Group IIA and IIC were derivedfrom inflammation-associated tissues (e.g., the synovium), and Group Vwere from cardiac tissue. The pancreatic PLA2s function in the digestionof dietary lipids and have been proposed to play a role in cellproliferation, smooth muscle contraction, and acute lung injury. TheGroup II inflammatory PLA2s are potent mediators of inflammatoryprocesses and are highly expressed in serum and synovial fluids ofpatients with inflammatory disorders. These Group II PLA2s are found inmost human ca types assayed and are expressed in diverse pathologicalprocesses such as septic shock, intestinal cancers, rheumatoidarthritis, and epidermal hyperplasia. A Group V PLA2 has been clonedfrom brain tissue and is strongly expressed in heart tissue. A humanPLA2 was recently cloned from fetal lung, and based on its structuralproperties, appears to be the first member of a new group of mammalianPLA2s, referred to as Group X. Other PLA2s have been cloned from varioushuman tissues and cell lies, suggest a large diversity of PLA2s (Chen,J. et al. (1994) J. Biol. Chem. 269:2365-2368; Kennedy, B. P. et al.(1995) J. Biol. Chem. 270: 22378-22385; Komada, M. et al. (1990)Biochem. Biophys. Res. Common 168:1059-1065; Cupillard, L. et al. (1997)J. Biol. Chem. 272:15745-15752; and Nalefski, E. A. et al. (1994) J.Biol. Chem. 269:18239-18249).

Phospholipases B (PLB) (ExPASy ENZYME EC 3.1.1.5), also known aslysophospholipase, lecithinase B, or lysolecithinase are widelydistributed enzymes that metabolize intracellular lipids, and occur innumerous isoforms. Small isoforms, approximately 15-30 kD, function ashydrolases; large isoforms, those exceeding 60 kD, function both ashydrolases and transacylases. A particular substrate for PLBs,lysophosphatidylcholine, causes lysis of cell membranes when it isformed or imported into a cell. PLBs are regulated by lipid factorsincluding acylcarnitine, arachidonic acid, and phosphatidic acid. Theselipid factors are signaling molecules important in numerous pathways,including the inflammatory response (Anderson, R. et al. (1994) Toxicol.Appl. Pharmacol. 125:176-183; Selle, H. et al. (1993); Eur. J. Biochem.212:4411-16).

Phospholipase C (PLC) (ExPASy ENZYME EC 3.1.4.10) plays an importantrole in transmembrane signal transduction. Many extracellular signalingmolecules including hormones, growth factors, neurotransmitters, andimmunoglobulins bind to their respective cell surface receptors andactivate PLCs. The role of an activated PLC is to catalyze thehydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP2), a minorcomponent of the plasma membrane, to produce diacylglycerol and inositol1,4,5-trisphosphate (IP3). In their respective biochemical pathways, IP3and diacylglycerol serve as second messengers and trigger a series ofintracellular responses. IP3 induces the release of Ca²⁺ from internalcellular storage, and diacylglycerol activates protein kinase C (PKC).Both pathways are part of transmembrane signal transduction mechanismswhich regulate cellular processes which include secretion, neuralactivity, metabolism, and proliferation.

Several distinct isoforms of PLC have been identified and arecategorized as PLC-beta, PLC-gamma, and PLC-delta. Subtypes aredesignated by adding Arabic numbers after the Greek letters, eg.PLC-β-1. PLCs have a molecular mass of 62-68 kDa, and their amino acidsequences show two regions of significant similarity. The first regiondesignated X has about 170 amino acids, and the second or Y regioncontains about 260 amino acids.

The catalytic activities of the three isoforms of PLC are dependent uponCa²⁺. It has been suggested that the binding sites for Ca²⁺ in the PLCsare located in the Y-region, one of two conserved regions. Thehydrolysis of common inositol-containing phospholipids, such asphosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP),and phosphatidylinositol 4,5-bisphosphate (PIP2), by any of the isoformsyields cyclic and noncyclic inositol phosphates (Rhee, S. G. and Y. S.Bae (1997) J. Biol. Chem. 272:15045-15048).

All mammalian PLCs contain a pleckstrin homology (PH) domain which isabout 100 amino acids in length and is composed of two antiparallel betasheets flanked by an amphipatic alpha helix. PH domains target PLCs tothe membrane surface by interacting with either the beta/gamma subunitsof G proteins or PIP2 (PROSITE PDOC50003).

Phospholipase D (PLD) (ExPASy ENZYME EC 3.1.4.4), also known aslecithinase D, lipophosphodiesterase II, and choline phosphatasecatalyzes the hydrolysis of phosphatidylcholine and other phospholipidsto generate phosphatidic add. PLD plays an important role in membranevesicle trafficking, cytoskeletal dynamics, and transmembrane signaltransduction. In addition, the activation of PLD is involved in celldifferentiation and growth (reviewed in Liscovitch, M. (2000) Biochem.J. 345:401-415).

PLD is activated in mammalian cells in response to diverse stimuli thatinclude hormones, neurotransmitters, growth factors, cytokines,activators of protein kinase C, and agonists binding toG-protein-coupled receptors. At least two forms of mammalian PLD, PLD1and PLD2, have been identified PLD1 is activated by protein kinase Calpha and by the small GTPases ARF and RhoA. (Houle, M. G. and S.Bourgoin (1999) Biochim. Biophys. Acta 1439:135-149). PLD2 can beselectively activated by unsaturated fatty acids such as oleate (Kim J.H. (1999) FEBS Lett. 454:42-46).

Lipoxygenases

Lipoxygenases (ExPASy ENZYME EC 1.13.11.12) are non-heme iron-containingenzymes that catalyze the dioxygenation of certain polyunsaturated fattyacids such as lipoproteins. Lipoxygenases are found widely in plants,fungi, and animals. Several different lipoxygenase enzymes are known,each having a characteristic oxidation action. In animals, there arespecific lipoxygenases that catalyze the dioxygenation of arachidonicacid at the carbon-3, 5, 8, 11, 12, and 15 positions. These enzymes arenamed after the position of arachidonic acid that they dioxygenate.Lipoxygenases have a single polypeptide chain with a molecular mass of˜75-80 kDa in animals. The proteins have an N-terminal-barrel domain anda larger catalytic domain containing a single atom of non-heme iron.Oxidation of the ferric enzyme to an active form is required forcatalysis (Yamamoto, S. (1992) Biochim. Biophys. Acta 1128:117-131;Brash, A. R. (1999) J. Biol. Chem. 274:23679-23682). A variety oflipoxygenase inhibitors exist and are classified into five majorcategories according to their mechanism of inhibition. These includeantioxidants, iron chelators, substrate analogues,lipoxygenase-activating protein inhibitors, and, finally, epidermalgrowth factor-receptor inhibitors.

3-Lipoxygenase, also known as e-LOX-3 or Aloxe3 has recently been clonedfrom murine epidermis. Aloxe3 resides on mouse chromosome 11, and thededuced amino acid sequence for Aloxe3 is 54% identical to the12-lipoxygenase sequences (Kinzig, A. (1999) Genomics 58:158-164).

5-Lipoxygenase (5-LOX, ExPASy ENZYME EC 1.13.11.34), also known asarachidonate:oxygen 5-oxidoreductase, is found primarily in white bloodcells, macrophages, and mast cells. 5-LOX converts arachidonic acidfirst to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and then toleukotriene (LTA4 (5,6-oxido-7,9,11,14-eicosatetraenoic acid)).Subsequent conversion of leukotriene A4 by leukotriene A4 hydrolaseyields the potent neutrophil chemoattractant leukotriene B4.Alternatively, conjugation of LTA4 with glutathione by leukotriene C4synthase plus downstream metabolism leads to the cysteinyl leukotrienesthat influence airway reactivity and mucus secretion, especially inasthmatics. Most lipoxygenases require no other cofactors or proteinsfor activity. In contrast, the mammalian 5-LOX requires calcium and ATP,and is activated in the presence of a 5-LOX activating protein (FLAP).FLAP itself binds to arachidonic acid and supplies 5-LOX with substrate(Lewis, R. A. et al. (1990) New Engl. J. Med. 323:645-655). Theexpression levels of 5-LOX and FLAP are found to be increased in thelungs of patients with plexogenic (primary) pulmonary hypertension(Wright, L. et al. (1998) Am. J. Respir. Crit. Care Med. 157:219-229).

12-Lipoxygenase (12-LOX, ExPASy ENZYME: EC 1.13.11.31) oxygenatesarachidonic acid to form 12-hydroperoxyeicosatetraenoic acid (12-HPETE).Mammalian 12-lipoxygenases are named after the prototypical tissues oftheir occurrence (hence, the leukocyte, platelet, or epidermal types).Platelet-type 12-LOX has been found to be the predominant isoform inepidermal skin specimens and epidermoid cells. Leukocyte 12-LOX wasfirst characterized extensively from porcine leukocytes and was found tohave a rather broad distribution in mammalian tissues by immunochemicalassays. Besides tissue distribution, the leukocyte 12-LOX isdistinguished from the platelet-type enzyme by its ability to form15-HPETE, in addition to 12-HPETE from arachidonic acid substrate.Leukocyte 12-LOX is highly related to 15-lipoxgenase (15-LOX) in thatboth are dual specificity lipoxygenases, and they are about 85%identical in primary structure in higher mammals. Leukocyte 12-LOX isfound in tracheal epithelium, leukocytes, and macrophages (Conrad, D. J.(1999) Clin. Rev. Allergy Immunol. 17:71-89).

15-Lipoxygenase (15-LOX; ExPASy ENZYME: EC 1.13.11.33) is found inhumanreticulocytes, airway epithelium, and eosinophils. 15-LOX has beendetected in atherosclerotic lesions in mammals, specifically rabbit andman. The enzyme, in addition to its role in oxidative modification oflipoproteins, is important in the inflammatory reaction inatherosclerotic lesions. 15-LOX has been shown to be induced in humanmonocytes by the cytokine IL-4, which is known to be implicated in theinflammatory process (Kuhn, H. and S. Borngraber (1999) Adv. Exp. Med.Biol. 447:5-28).

Disease Correlation

Lipid metabolism is involved in human diseases and disorders. In thearterial disease atherosclerosis, fatty lesions form on the inside ofthe atrial wall. These lesions promote the loss of arterial flexibilityand the formation of blood clots (Guyton, supra). In Tay-Sachs disease,the GM₂ ganglioside (a sphingolipid) accumulates in lysosomes of thecentral nervous system due to a lack of the enzymeN-acetylhexosaminidase. Patients suffer nervous system degenerationleading to early death (Fauci, A. S. et al. (1998) Harrison's Principlesof Internal Medicine, McGraw-Hill, New York N.Y., p. 2171). TheNiemann-Pick diseases are caused by defects in lipid metabolism.Niemann-Pick diseases types A and B are caused by accumulation ofsphingomyelin (a sphingolipid) and or lipids in the central nervoussystem due to a defect in the enzyme sphingomyelinase, leading toneurodegeneration and lung disease. Niemann-Pick disease type C resultsfrom a defect in cholesterol transport, leading to the accumulation ofsphingomyelin and cholesterol in lysosomes and a secondary reduction insphingomyelinase activity. Neurological symptoms such as grand malseizures, ataxia, and loss of previously learned speech, manifest 1-2years after birth. A mutation in the NPC protein, which contains aputative cholesterol-sensing domain, was found in a mouse model ofNiemann-Pick disease type C (Fauci, supra, p. 2175; Loftus, S. K. et al.(1997) Science 277:232-235).

PLAs are implicated in a variety of disease processes. For example, PLAsare found in the pancreas, in cardiac tissue, and ininflammation-associated tissues. Pancreatic PLAs function in thedigestion of dietary lipids and have been propose to play a role in cellproliferation, smooth muscle contraction, and acute lung injury.Inflammatory PLAs are potent mediators of inflammatory processes and arehighly expressed in serum and synovial fluids of patients withinflammatory disorders. Additionally, inflammatory PLAs are found inmost human cell types and are expressed in diverse pathologicalprocesses such as septic shock, intestinal cancers, rheumatoidarthritis, and epidermal hyperplasia.

The role of PLBs in human tissues has been investigated in variousresearch studies. Hydrolysis of lysophosphatidylcholine by PLBs causeslysis in erythrocyte membranes (Selle, supra). Similarly, Endresen, M.J. et al. (1993; Scand. J. Clin. Invest. 53:733-739) reported that theincreased hydrolysis of lysophosphatidylcholine by PLB in pre-eclampticwomen causes release of free fatty acids into the sera. In renalstudies, PLB was shown to protect Na⁺,K⁺-ATPase from the cytotoxic andcytolytic effects of cyclosporin A (Anderson, supra).

Lipases, phospholipases, and lipoxygenases are thought to contribute tocomplex diseases, such as atherosclerosis, obesity, arthritis, asthma,and cancer, as well as to single gene defects, such as Wolman's diseaseand Type I hyperlipoproteinemia.

The discovery of new lipid metabolism enzymes and the polynucleotidesencoding them satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention, and treatment of cancer,neurological disorders, autoimmune/inflammatory disorders,gastrointestinal disorders, and cardiovascular disorders, and in theassessment of the effects of exogenous compounds on the expression ofnucleic acid and amino acid sequences of lipid metabolism enzymes.

SUMMARY OF THE INVENTION

The invention features purified polypeptides, lipid metabolism enzymes,referred to collectively as “LME” and individually as “LME-1,” “LME-2,”“LME-3,” “LME-4,” “LME-5,” and “LME-6.” In one aspect, the inventionprovides an isolated polypeptide comprising an amino acid sequenceselected from the group consisting of a) an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-6, b) a naturally occurringamino acid sequence having at least 90% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NO:1-6, c) abiologically active fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of anamino acid sequence selected from the group consisting of SEQ ID NO:1-6.In one alternative, the invention provides an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:1-6.

The invention further provides an isolated polynucleotide encoding apolypeptide comprising an amino acid sequence selected from the groupconsisting of a) an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, c) a biologicallyactive fragment of amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-6. Inone alternative, the polynucleotide encodes a polypeptide selected fromthe group consisting of SEQ ID NO:1-6. In another alternative, thepolynucleotide is selected from the group consisting of SEQ ID NO:7-12.

Additionally, the invention provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromthe group consisting of a) an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-6, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, c) a biologicallyactive fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-6. Inone alternative, the invention provides a cell transformed with therecombinant polynucleotide. In another alternative, the inventionprovides a transgenic organism comprising the recombinantpolynucleotide.

The invention also provides a method for producing a polypeptidecomprising an amino acid sequence selected from the group consisting ofa) an amino acid sequence selected from the group consisting of SEQ IDNO:1-6, b) a naturally occurring amino acid sequence having at least 90%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6, c) a biologically active fragment of anamino acid sequence selected from the group consisting of SEQ ID NO:1-6,and d) an immunogenic fragment of an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-6. The method comprises a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding the polypeptide, and b) recovering the polypeptide soexpressed.

Additionally, the invention provides an isolated antibody whichspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of a) an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-6, b) a naturally occurringamino acid sequence having at least 90% sequence identity to a aminoacid sequence selected from the group consisting of SEQ ID NO:1-6, c) abiologically active fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of anamino acid sequence selected from the group consisting of SEQ ID NO:1-6.

The invention further provides an isolated polynucleotide comprising apolynucleotide sequence selected from the group consisting of a) apolynucleotide sequence selected from the group consisting of SEQ IDNO:7-12, b) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:7-12, c) a polynucleotide sequencecomplementary to a), d) a polynucleotide sequence complementary to b),and e) an RNA equivalent of a)-d). In one alternative, thepolynucleotide comprises at least 60 contiguous nucleotides.

Additionally, the invention provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of a) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:7-12, b) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:7-12, c) a polynucleotide sequence complementary to a), d) apolynucleotide sequence complementary to b), and e) an RNA equivalent ofa)-d). The method comprises a) hybridizing the sample with a probecomprising at least 20 contiguous nucleotides comprising a sequencecomplementary to said target polynucleotide in the sample, and whichprobe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. In one alternative, the probe comprisesat least 60 contiguous nucleotides.

The invention further provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of a) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:7-12, b) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:7-12, c) a polynucleotide sequence complementary to a), d) apolynucleotide sequence complementary to b), and e) an RNA equivalent ofa)-d). The method comprises a) amplifying said target polynucleotide orfragment thereof using polymerase chain reaction amplification, and b)detecting the presence or absence of said amplified targetpolynucleotide or fragment thereof, and, optionally, if present, theamount thereof.

The invention further provides a composition comprising an effectiveamount of a polypeptide comprising an amino acid sequence selected fromthe group consisting of a) an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-6, b) a naturally occurring amino acidsequence having at least 90% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, c) a biologicallyactive fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6, and d) an immunogenic fragment of an aminoacid sequence selected from the group consisting of SEQ ID NO:1-6, and apharmaceutically acceptable excipient. In one embodiment, thecomposition comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6. The invention additionally provides amethod of treating a disease or condition associated with decreasedexpression of functional LME, comprising administering to a patient inneed of such treatment the composition.

The invention also provides a method for screening a compound foreffectiveness as an agonist of a polypeptide comprising an amino acidsequence selected from the group consisting of a) an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, b) a naturallyoccurring amino acid sequence having at least 90% sequence identity toan amino acid sequence selected from the group consisting of SEQ IDNO:1-6, c) a biologically active fragment of an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, and d) animmunogenic fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6. The method comprises a) exposing a samplecomprising the polypeptide to a compound, and b) detecting agonistactivity in the sample. In one alternative, the invention provides acomposition comprising an agonist compound identified by the method anda pharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with decreased expression of functional LME, comprisingadministering to a patient in need of such treatment the composition.

Additionally, the invention provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide comprising an aminoacid sequence selected from the group consisting of a) an amino acidsequence selected from the group consisting of SEQ ID NO:1-6, b) anaturally occurring amino acid sequence having at least 90% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:1-6, c) a biologically active fragment of an amino acidsequence selected from the group consisting of SEQ ID NO:1-6, and d) animmunogenic fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6. The method comprises a) exposing a samplecomprising the polypeptide to a compound, and b) detecting antagonistactivity in the sample. In one alternative, the invention provides acomposition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. In another alternative, theinvention provides a method of treating a disease or conditionassociated with overexpression of functional LME, comprisingadministering to a patient in need of such treatment the composition.

The invention further provides a method of screening for a compound thatspecifically binds to a polypeptide comprising an amino acid sequenceselected from the group consisting of a) an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-6, b) a naturally occurringamino acid sequence having at least 90% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NO:1-6, c) abiologically active fragment of an ammo acid sequence selected from thegroup consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of anamino acid sequence selected from the group consisting of SEQ ID NO:1-6.The method comprises a) combining the polypeptide with at least one testcompound under suitable conditions, and b) detecting binding of thepolypeptide to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide.

The invention further provides a method of screening for a compound thatmodulates the activity of a polypeptide comprising an amino acidsequence selected from the group consisting of a) an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, b) a naturallyoccurring amino acid sequence having at least 90% sequence identity toan amino acid sequence selected from the group consisting of SEQ IDNO:1-6, c) a biologically active fragment of an amino acid sequenceselected from the group consisting of SEQ ID NO:1-6, and d) animmunogenic fragment of an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-6. The method comprises a) combining thepolypeptide with at least one test compound under conditions permissivefor the activity of the polypeptide, b) assessing the activity of thepolypeptide in the presence of the test compound, and c) comparing theactivity of the polypeptide in the presence of the test compound withthe activity of the polypeptide in the absence of the test compound,wherein a change in the activity of the polypeptide in the presence ofthe test compound is indicative of a compound that modulates theactivity of the polypeptide.

The invention further provides a method for screening a compound foreffectiveness in altering expression of a target polynucleotide, whereinsaid target polynucleotide comprises a sequence selected from the groupconsisting of SEQ ID NO:7-12, the method comprising a) exposing a samplecomprising the target polynucleotide to a compound, and b) detectingaltered expression of the target polynucleotide.

The invention further provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide comprising apolynucleotide sequence selected from the group consisting of i) apolynucleotide sequence selected from the group consisting of SEQ IDNO:7-12, ii) a naturally occurring polynucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:7-12, iii) a polynucleotide sequencecomplementary to i), iv) a polynucleotide sequence complementary to ii),and v) an RNA equivalent of i)-iv). Hybridization occurs underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence selected fromthe group consisting of i) a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:7-12, ii) a naturally occurringpolynucleotide sequence having at least 90% sequence identity to apolynucleotide sequence selected from the group consisting of SEQ IDNO:7-12, ii) a polynucleotide sequence complementary to i), iv) apolynucleotide sequence complementary to ii), and v) an RNA equivalentof i)-iv). Alternatively, the target polynucleotide comprises a fragmentof a polynucleotide sequence selected from the group consisting of i)-v)above; c) quantifying the amount of hybridization complex; and d)comparing the amount of hybridization complex in the treated biologicalsample with the amount of hybridization complex in an untreatedbiological sample, wherein a difference in the amount of hybridizationcomplex in the treated biological sample is indicative of toxicity ofthe test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the present invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog for polypeptides of the invention. Theprobability score for the match between each polypeptide and its GenBankhomolog is also shown.

Table 3 shows structural features of polypeptide sequences of theinvention, including predicted motifs and domains, along with themethods, algorithms, and searchable databases used for analysis of thepolypeptides.

Table 4 lists the cDNA fragments which were used to assemblepolynucleotide sequences of the invention, along with selected fragmentsof the polynucleotide sequences.

Table 5 shows the representative cDNA library for polynucleotides of theinvention.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze thepolynucleotides and polypeptides of the invention, along with applicabledescriptions, references, and threshold parameters.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Definitions

“LME” refers to the amino acid sequences of substantially purified LMEobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine, and human, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of LME. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of LME either by directlyinteracting with LME or by acting on components of the biologicalpathway in which LME participates.

A “allelic variant” is an alternative form of the gene encoding LME.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding LME include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as LME or a polypeptide with atleast one functional characteristic of LME. Included with thisdefinition are polymorphisms which may or may not be readily detectableusing a particular of oligonucleotide probe of the polynucleotideencoding LME, and improper or unexpected hybridization to allelicvariants, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding LME. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent LME. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphiphatic nature of theresidues, as long as the biological or immunological activity of LME isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, and positively charged amino acids mayinclude lysine and arginine. Amino acids with uncharged polar sidechains having similar hydrophilicity values may include: asparagine andglutamine; and serine and threonine. Amino acids with uncharged sidechains having similar hydrophilicity values may include: leucine,isoleucine, and valine; glycine and alanine; and phenylalanine andtyrosine.

The terms “amino acid” and “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.Where “amino acid sequence” is recited to refer to a sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not mean to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of LME. Antagonists may include proteins such asantibodies, nucleic acids, carbohydrates, small molecules, or any othercompound or composition which modulates the activity of LME either bydirectly interacting with LME or by acting on components of thebiological pathway in which LME participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind LMEpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete wt the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “antisense” refers to any composition capable of base-paringwith we “sense” (coding) stand of a specific nucleic acid sequence.Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA);oligonucleotides having modified backbone linkages such asphosphorothioates, methylphosphonates, or benzylphosphonates;oligonucleotides having modified sugar groups such as 2′-methoxyethylsugars or 2′-methoxyethoxy sugars; or oligonucleotides having modifiedbases such as 5-methyl cytosine, 2′-deoxyuracil, or7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by anymethod including chemical synthesis or transcription. Once introducedinto a cell, the complementary antisense molecule base-pairs with anaturally occurring nucleic acid sequence produced by the cell to formduplexes which block either transcription or translation. Thedesignation “negative” or “minus” can refer to the antisense strand, andthe designation “positive” or “plus” can refer to the sense strand of areference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic LME, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide sequence” and a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encoding LMEor fragments of LME may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate, SDS), and other components(e.g., Denhardt's solution, dry milk salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genomic DNAfragments using a computer program for fragment assembly, such as theGEL VIEW fragment assembly system (GCG, Madison Wis.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredict to least interfere with the properties of the original proteini.e., the structure and especially the function of the protein isconserved and not significantly changed by such substitutions. The tableblow shows amino acids which may be substituted for an original aminoacid in a protein and which are regarded as conservative amino acidsubstitutions.

Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys AsnAsp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln,His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg,Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser,Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino add or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

A “fragment” is a unique portion of LME or the polynucleotide encodingLYE which is identical in sequence to but shorter in length than theparent sequence. A fragment may comprise up to the entire length of thedefined sequence, minus one nucleotide/amino acid residue. For example,a fragment may comprise from 5 to 1000 contiguous nucleotides or aminoacid residues. A fragment used as a probe, primer, antigen, therapeuticmolecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25,30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotidesor amino acid residues in length. Fragments may be preferentiallyselected from certain regions of a molecule. For example, a polypeptidefragment may comprise a certain length of contiguous amino acidsselected from the first 250 or 500 amino acids (or first 25% or 50%) ofa polypeptide as shown in a certain defined sequence. Clearly theselengths are exemplary, and any length that is supported by thespecification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.

A fragment of SEQ ID NO:7-12 comprises a region of unique polynucleotidesequence that specifically identifies SEQ ID NO:7-12, for example, asdistinct from any other sequence in the genome from which the fragmentwas obtained. A fragment of SEQ ID NO:7-12 is useful, for example, inhybridization and amplification technologies and in analogous methodsthat distinguish SEQ ID NO:7-12 from related polynucleotide sequences.The precise length of a fragment of SEQ ID NO:7-12 and the region of SEQID NO:7-12 to which the fragment corresponds are routinely determinableby one of ordinary skill in the art based on the intended purpose forthe fragment.

A fragment of SEQ ID NO:1-6 is encoded by a fragment of SEQ ID NO:7-12,A fragment of SEQ ID NO:1-6 comprises a region of unique amino acidsequence that specifically identifies SEQ ID NO:1-6. For example, afragment of SEQ ID NO:1-6 is useful as an immunogenic peptide for thedevelopment of antibodies that specifically recognize SEQ ID NO:1-6. Theprecise length of a fragment of SEQ ID NO; 1-6 and the region of SEQ IDNO:1-6 to which the fragment corresponds are routinely determinable byone of ordinary skill in the at based on the intended purpose for thefragment.

A “full length” polynucleotide sequence is one containing at least atranslation initiation codon (e.g., methionine) followed by an openreading frame and a translation termination codon. A “full length”polynucleotide sequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, interchangeably, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of residue matchesbetween at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing the default parameters of the CLUSTAL V algorithm as incorporatedinto the MEGALIGN version 3.12e sequence alignment program. This programis part of the LASERGENE software package, a suite of molecularbiological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V isdescribed in Higgins, D. G. and P. M. Sharp (1989) CABIOS 5:151-153 andin Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwisealignments of polynucleotide sequences, the default parameters are setas follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4.The “weighted” residue weight table is selected as the default. Percentidentity is reported by CLUSTAL V as the “percent similarity” betweenaligned polynucleotide sequences.

Alernatively, a suite of commonly used and freely available sequencecomparison algorithms is provided by the National Center forBiotechnology Information (NCBI) Basic Local Alignment Search Tool(BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 2 15:403-4 10),which is available from several sources, including the NCBI, Bethesda,Md., and on the Internet. The BLAST software suite includes varioussequence analysis programs including “blastn,” that is used to align aknown polynucleotide sequence with other polynucleotide sequences from avariety of databases. Also available is a tool called “BLAST 2Sequences” that is used for direct pairwise comparison of two nucleotidesequences. “BLAST 2 Sequences” can be accessed and used interactively onthe internet. The “BLAST 2 Sequences” tool can be used for both blastnand blastp (discussed below). BLAST programs are commonly used with gapand other parameters set to default settings. For example, to comparetwo nucleotide sequences, one may use blastn with the “BLAST 2Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at defaultparameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: −2

Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50

Expect 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof ale genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table. As with polynucleotidealignments, the percent identity is reported by CLUSTAL V as the“percent similarity” between aligned polypeptide sequence pairs.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp setat default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap x drop-off: 50

Expect: 10

Word Size: 3

Filter: on

Percent identity may be measured over the length of an entire defiedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defied polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and shill retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. et al. (1989) Molecular Cloning:A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; specifically see volume 2, chapter 9.

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions millbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀t or R₀t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” and “addition” refer to changes in an amino acidor nucleotide sequence resulting in the addition of one or more aminoacid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofLME which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment of LMEwhich is useful in any of the antibody production methods disclosedherein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, or other chemical compounds on asubstrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, or other chemical compound having a unique and definedposition on a microarray.

The term “modulate” refers to a change in the activity of LME. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of LME.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense stand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an LME may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of LME.

“Probe” refers to nucleic acid sequences encoding LME, theircomplements, or fragments thereof, which are used to detect identical,allelic or related nucleic acid sequences. Probes are isolatedoligonucleotides or polynucleotides attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. “Primers” are short nucleic acids,usually DNA oligonucleotides, which may be annealed to a targetpolynucleotide by complementary base-pairing. The primer may then beextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification (and identification) of a nucleicacid sequence, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed suchas probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70,80, 90, 100, or at least 150 consecutive nucleotides of the disclosednucleic acid sequences. Probes and primers may be considerably longerthan these examples, and it is understood that any length supported bythe specification, including the tables, figures, and Sequence Listing,may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook, supra. The term recombinant includes nucleic acids thathave been altered solely by addition, substitution, or deletion of aportion of the nucleic acid. Frequently, a recombinant nucleic acid mayinclude a nucleic acid sequence operably linked to a promoter sequence.Such a recombinant nucleic acid may be part of a vector that is used,for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector e.g., based on a vaccinia virus, that could be use to vaccinate amammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′, untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA sequence, is composed of thesame linear sequence of nucleotides as the reference DNA sequence withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining LME, nucleic acids encoding LME, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least 60% free, preferably at least75% free, and most preferably at least 90% free from other componentswith which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” refers to the collective pattern of gene expressionby a particular cell type or tissue under given conditions at a giventime.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.The transgenic organisms contemplated in accordance with the presentinvention include bacteria, cyanobacteria, fungi, plants and animals.The isolated DNA of the present invention can be introduced into thehost by methods known in the art, for example infection, transfection,transformation or transconjugation. Techniques for transferring the DNAof the present invention into such organisms are widely known andprovided in references such as Sambrook et al. (1989), supra.

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotide sequences that vary fromone species to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity to theparticular polypeptide sequence over a certain length of one of thepolypeptide sequences using blastp with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofpolypeptides may show, for example, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% or greater sequence identity over a certain definedlength of one of the polypeptides.

The Invention

The invention is based on the discovery of new human lipid metabolismenzymes (LME), the polynucleotides encoding LME, and the use of thesecompositions for the diagnosis, treatment, or prevention of cancer,neurological disorders, autoimmune/inflammatory disorders,gastrointestinal disorders, and cardiovascular disorders.

Table 1 summarizes the nomenclature for the full length polynucleotideand polypeptide sequences of the invention. Each polynucleotide and itscorresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Bach polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown.

Table 2 shows sequences with homology to the polypeptides of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database. Columns 1 and 2 show the polypeptide sequenceidentification number (Polypeptide SEQ ID NO:) and the correspondingIncyte polypeptide sequence number (Incyte Polypeptide ID) forpolypeptides of the invention. Column 3 shows the GenBank identificationnumber (Genbank ID NO:) of the nearest GenBank homolog. Column 4 showsthe probability score for the match between each polypeptide and itsGenBank homolog. Column 5 shows the annotation of the GenBank homologalong with relevant citations where applicable, all of which areexpressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of ammo acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Genetics Computer Group,Madison Wis.). Column 6 shows amino acid residues comprising signaturesequences, domains, and motifs. Column 7 shows analytical methods forprotein structure/function analysis and in some cases, searchabledatabases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare lipid metabolism enzymes. For example, SEQ ID NO:1 is 50% identical,from residue N63 to residue H300, to a C. elegans protein havingsimilarity to human enoyl-CoA hydratase (GenBank ID g3876901) asdetermined by the Basic Local Alignment Search Tool (BLAST). (See Table2.) The BLAST probability score is 2.50E-56, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:1 also contains an enoyl-CoA hydratase/isomerasefamily domain as determined by searching for statistically significantmatches in the hidden Markov model (HMM)-based PFAM database ofconserved protein family domains. (See Table 3.) Data from BLIMPS andPROFILESCAN analyses provide further corroborative evidence that SEQ IDNO:1 is an enoyl-CoA hydratase. SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, and SEQ ID NO:6 were analyzed and annotated in a similarmanner. The algorithms and parameters for the analysis of SEQ ID NO:1-6are described in Table 7.

As shown in Table 4, the full length polynucleotide sequences of thepresent invention were assembled using cDNA sequences or coding (exon)sequences derived from genomic DNA, or any combination of these twotypes of sequences. Columns 1 and 2 list the polynucleotide sequenceidentification number (Polynucleotide SEQ ID NO:) and the correspondingIncyte polynucleotide consensus sequence number (Incyte PolynucleotideID) for each polynucleotide of the invention. Column 3 shows the lengthof each polynucleotide sequence in basepairs. Column 4 lists fragmentsof the polynucleotide sequences which are useful, for example, inhybridization or amplification technologies that identify SEQ ID NO:7-12or that distinguish between SEQ ID NO:7-12 and related polynucleotidesequences. Column 5 shows identification numbers corresponding to cDNAsequences, coding sequences (exons) predicted from genomic DNA, and/orsequence assemblages comprised of both cDNA and genomic DNA. Thesesequences were used to assemble the full length polynucleotide sequencesof the invention. Columns 6 and 7 of Table 4 show the nucleotide start(5′) and stop (3′) positions of the cDNA sequences in column 5 relativeto their respective full length sequences.

The identification numbers in Column 5 of Table 4 may referspecifically, for example, to Incyte cDNAs along with theircorresponding cDNA libraries. For example, 6827519J1 is theidentification number of an Incyte cDNA sequence, and SINTNOR01 is thecDNA library from which it is derived. Incyte cDNAs for which cDNAlibraries are not indicated were derived from pooled cDNA libraries(e.g., 71530085V1). Alternatively, the identification numbers in column5 may refer to GenBank cDNAs or ESTs which contributed to the assemblyof the fall length polynucleotide sequences. Alternatively, theidentification numbers in column 5 may refer to coding regions predictedby Genscan analysis of genomic DNA. The Genscan-predicted codingsequences may have been edited prior to assembly. (See Example IV.)Alternatively, the identification numbers in column 5 may refer toassemblages of both cDNA and Genscan-predicted exons brought together byan “exon stitching” algorithm. (See Example V.) Alternatively, theidentification numbers in column 5 may refer to assemblages of both cDNAand Genscan-predicted exons brought together by an “exon-stretching”algorithm. (See Example V.) In some cases, Incyte cDNA coverageredundant with the sequence coverage shown in column 5 was obtained toconfirm the final consensus polynucleotide sequence, but the relevantIncyte cDNA identification numbers are not shown.

Table 5 shows the representative cDNA libraries for those fall lengthpolynucleotide sequences which were assembled using Incyte cDNAsequences. The representative cDNA library is the Incyte cDNA librarywhich is most frequently represented by the Incyte cDNA sequences whichwere used to assemble and confirm the above polynucleotide sequences.The tissues and vectors which were used to construct the cDNA librariesshown in Table 5 are described in Table 6.

The invention also encompasses LME variants. A preferred LME variant isone which has at least about 80%, or alternatively at least about 90%,or even at least about 95% amino acid sequence identity to the LME aminoacid sequence, and which contains at least one functional or structuralcharacteristic of LME.

The invention also encompasses polynucleotides which encode LME. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:7-12, which encodes LME. The polynucleotide sequences of SEQ IDNO:7-12, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses a variant of a polynucleotide sequenceencoding LME. In particular, such a variant polynucleotide sequence willhave at least about 70%, or alternatively at least about 85%, or even atleast about 95% polynucleotide sequence identity to the polynucleotidesequence encoding LME. A particular aspect of the invention encompassesa variant of a polynucleotide sequence comprising a sequence selectedfrom the group consisting of SEQ ID NO:7-12 which has at least about70%, or alternatively at least about 85%, or even at least about 95%polynucleotide sequence identity to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:7-12. Any one of thepolynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of LME.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude or polynucleotidesequences encoding LME, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring LME, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode LME and its variants aregenerally capable of hybridizing to the nucleotide sequence of thenaturally occurring LME under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding LME or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Otter reasons for substantially altering the nucleotide sequenceencoding LME and its derivatives without altering the encoded amino acidsequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeLME and LME derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and all systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding LME or any fragmentthereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:7-12 and fragments thereof undervarious conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) MethodsEnzymol. 152:507-511.) Hybridization conditions, including annealing andwash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the MICROLAB 2200 liquid transfer system (Hamilton,Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABICATALYST 800 thermal cycler (Applied Biosystems). Sequencing is thencarried out using either the ABI 373 or 377 DNA sequencing system(Applied Biosystems), the MEGABACE 1000 DNA sequencing system (MolecularDynamics, Sunnyvale Calif.), or other systems known in the art. Theresulting sequences are analyzed using a variety of algorithms which arewell known in the art. (See, e.g., Ausubel, F. M. (1997) Short Protocolsin Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7;Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, NewYork N.Y., pp. 856-853.)

The nucleic acid sequences encoding LME may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extended in divergent directions toamplify unknown sequence from a circularized template. The template isderived from restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode LME may be cloned in recombinant DNAmolecules that direct expression of LME, or fragments or functionalequivalents thereof, in appropriate host cells. Due to tee inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express LME.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter LME-encodingsequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of LME, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, sequences encoding LME may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223;and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.)Alternatively, LME itself or a fragment thereof may be synthesized usingchemical methods. For example, peptide synthesis can be performed usingvarious solution-phase or solid-phase techniques. (See, e.g., Creighton,T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, NewYork N.Y., pp. 55-60; and Roberge, J. Y. et al. (1995) Science269:202-204.) Automated synthesis may be achieved using the ABI 431Apeptide synthesizer (Applied Biosystems). Additionally, the amino acidsequence of LME, or any part thereof may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide or a polypeptide having asequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, supra, pp. 28-53.)

In order to express a biologically active LME, the nucleotide sequencesencoding LME or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesequences encoding LME. Such elements may vary in their strength andspecificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding LME. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding LME and its initiation codonand upstream regulatory sequences are inserted into the appropriateexpression vector, no additional transcriptional or translationalcontrol signals may be needed. However, in cases where only codingsequence, or a fragment thereof, is inserted, exogenous translationalcontrol signals including an in-frame ATG initiation codon should beprovided by the vector. Exogenous translational elements and initiationcodons may be of various origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of enhancersappropriate for the particular host cell system used. (See, e.g.,Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding LME andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination (See e.g., Sambrook, J. etal. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding LME. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See,e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J, and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; and Harrington, J. J. et al. (1997 Nat. Genet.15:345-355.) Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of nucleotide sequences to the targeted organ,tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998)Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad.Sci. USA 90(13):634-6344; Buller, R. M. et al. (1985) Nature317(6040):813-815; McGregor, D. P. et al. (1994) Mol. Immunol.31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature 389:239-242.)The invention is not limited by file host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding LME. For example, routine cloning, subcloning, and propagationof polynucleotide sequences encoding LME can be achieved using amultifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JollaCalif.) or PSPORT1 plasmid (Life Technologies). Ligation of sequencesencoding LME into the vector's multiple cloning site disrupts the lacZgene, allowing a colorimetric screening procedure for identification oftransformed bacteria containing recombinant molecules. In addition,these vectors may be useful for in vitro transcription, dideoxysequencing, single strand rescue with helper phage, and creation ofnested deletions in the cloned sequence. (See, e.g., Van Heeke, G. andS. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of LME are needed e.g. for the production of antibodies,vectors which direct high level expression of LME may be used. Forexample, vectors containing the strong, inducible SP6 or T7bacteriophage promoter may be used.

Yeast expression systems may be used for production of LME. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign sequences into the hostgenome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. etal. (1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of LME. Transcription ofsequences encoding LME may be driven by viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g. Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection (See, e.g., The McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New YorkN.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding LME may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective us which expressesLME in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl.Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, suchas the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells. SV40 or EBV-based vectors may alsobe used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat. Genet. 15:345-355.)

For long term production of recombinant proteins in mammalian systems,stable expression of LME in cell lines is preferred. For example,sequences encoding LME can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purse of the selectable marker isto confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ and apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin,F. et al. (1981) J. Mol. Biol. 150:1-14.) Addition selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP; Clontech), βglucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system.(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encoding LMEis inserted within a marker gene sequence, transformed cells containingsequences encoding LME can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding LME under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingLME and that express LME may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification,and protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of LMEusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-biked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sort (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on LME ispreferred, but a competitive binding assay may be employed. These andother assays are well known in the art. (See, e.g., Hampton, R. et al.(1990) Serological Methods, a Laboratory Manual, APS Press, St. PaulMinn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols inImmunology, Greene Pub. Associates and Wiley-Interscience, New YorkN.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press,Totowa N.J.)

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding LME includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding LME,or any fragments thereof may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitablereporter molecules or labels which may be used for ease of detectinginclude radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding LME may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeLME may be designed to contain signal sequences which direct secretionof LME through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding LME may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric LMEprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of LME activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the LME encodingsequence and the heterologous protein sequence, so that LME may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch. 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeled LMEmay be achieved in vitro using the TNT rabbit reticulocyte lysate orwheat germ extract system (Promega). These systems couple transcriptionand translation of protein-coding sequences operably associated with theT7, T3, or SP6 promoters. Translation takes place in the presence of aradiolabeled amino acid precursor, for example, ³⁵S-methionine.

LME of the present invention or fragments thereof may be used to screenfor compounds that specifically bind to LME. At least one and up to aplurality of test compounds may be screened for specific binding to LME.Examples of test compounds include antibodies, oligonucleotides,proteins (e.g., receptors), or small molecules.

In one embodiment, the compound thus identified is closely related tothe natural ligand of LME, e.g., a g, and or fragment thereof a naturalsubstrate, a structural or functional mimetic, or a natural bindingpartner, (See, e.g., Coligan, J. E. et al. (1991) Current Protocols inImmunology 1(2): Chapter 5.) Similarly, the compound can be closelyrelated to the natural receptor to which LME binds, or to at least afragment of the receptor, e.g., the ligand binding site. In either case,the compound can be rationally designed using known techniques. In oneembodiment, screening for these compounds involves producing appropriatecells which express LME, either as a secreted protein or on the cellmembrane. Preferred cells include cells from mammals, yeast, Drosophila,or E. coli. Cells expressing LME or cell membrane fractions whichcontain LME are then contacted with a test compound and binding,stimulation, or inhibition of activity of either LME or the compound isanalyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with LME,either in solution or affixed to a solid support, and detecting thebinding of LME to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

LME of the present invention or fragments thereof may be used to screenfor compounds that modulate the activity of LME. Such compounds mayinclude agonists, antagonists, or partial or inverse agonists. In oneembodiment, an assay is performed under conditions permissive for LMEactivity, wherein LME is combined with at least one test compound, andthe activity of LME in the presence of a test compound is compared withthe activity of LME in the absence of the test compound. A change in theactivity of LME in the presence of the test compound is indicative of acompound that modulates the activity of LME. Alternatively, a testcompound is combined with an in vitro or cell-free system comprising LMEunder conditions suitable for LME activity, and the assay is performed.In either of these assays, a test compound which modulates the activityof LME may do so indirectly and need not come in direct contact with thetest compound. At least one and up to a plurality of test compounds maybe screened.

In another embodiment, polynucleotides encoding LME or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycinphosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding LME may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding LME can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding LME is injected into animal ES cells, and the injected sequenceintegrates into the animal cell genome. Transformed cells are injectedinto blastulae, and the blastulae are implanted as described above.Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress LME, e.g.,by secreting LME in its milk, may also serve as a convenient source ofthat protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of LME and lipid metabolism enzymes.In addition, the expression of LME is closely associated withreproductive tissues, reproductive disorders, cancer, and therepresentative libraries listed in Table 6. Therefore, LME appears toplay a role in cancer, neurological disorders, autoimmune/inflammatorydisorders, gastrointestinal disorders, and cardiovascular disorders. Inthe treatment of disorders associated with increased LME expression oractivity, it is desirable to decrease the expression or activity of LME.In the treatment of disorders associated with decreased LME expressionor activity, it is desirable to increase the expression or activity ofLME.

Therefore, in one, embodiment, LME or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of LME. Examples ofsuch disorders include, but are not limited to, a cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brat, breast cervix, gall bladder, ganglia,gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen,testis, thymus, thyroid, and uterus; a neurological disorder such asepilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,Alzheimer's disease, Pick's disease, Huntington's disease, dementia,Parkinson's disease and other extrapyramidal disorders, amyotrophiclateral sclerosis and other motor neuron disorders, progressive neuralmuscular atrophy, retinitis pigmentosa, hereditary ataxias, multiplesclerosis and other demyelinating diseases, bacterial and viralmeningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; an autoimmune inflammatory disordersuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; agastrointestinal disorder such as dysphagia, peptic esophagitis,esophageal spasm, esophageal structure, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquiredimmunodeficiency syndrome (AIDS) enteropathy, jaundice, hepaticencephalopathy, hepatorenal syndrome, hepatic steatosis,hemochromatosis, Wilson's disease, alpha₁-antitrypsin deficiency, Reye'ssyndrome, primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, centrilobular necrosis, peliosis hepatis,hepatic vein thrombosis, veno-occlusive disease, preeclampsia,eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, and hepatic tumors including nodular hyperplasias, adenomas,and carcinomas; and a cardiovascular disorder such as arteriovenousfistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease,aneurysms, arterial dissections, varicose veins, thrombophlebitis andphlebothrombosis, vascular tumors, and complications of thrombolysis,balloon angioplasty, vascular replacement, and coronary artery bypassgraft surgery, congestive heart failure, ischemic heart disease, anginapectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, congenital heartdisease, and complications of cardiac transplantation, congenital lunganomalies, atelectasis, pulmonary congestion and edema, pulmonaryembolism, pulmonary hemorrhage, pulmonary infarction, pulmonaryhypertension, vascular sclerosis, obstructive pulmonary disease,restrictive pulmonary disease, chronic obstructive pulmonary disease,emphysema, chronic bronchitis, bronchial asthma, bronchiectasis,bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess,pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiliabronchiolitis obliterans-organizing pneumonia, diffuse pulmonaryhemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonaryhemosiderosis, pulmonary involvement in collagen-vascular disorders,pulmonary alveolar proteinosis, lung tumors, inflammatory andnoninflammatory pleural effusions, pneumothorax, pleural tumors,drug-induced lung disease, radiation-induced lung disease, andcomplications of lung transplantation.

In another embodiment, a vector capable of expressing LME or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofLME including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified LME in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of LME including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofLME may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of LME including, butnot limited to, those listed above.

In a further embodiment, an antagonist of LME may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of LME. Examples of such disorders include, butare not limited to, those cancers, neurological disorders,autoimmune/inflammatory disorders, gastrointestinal disorders, andcardiovascular disorders described above. In one aspect, an antibodywhich specifically binds LME may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissues which express LME.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding LME may be administered to a subject to treat orprevent a disorder associated with increased expression or activity ofLME including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of ft invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of LME may be produced using methods which are generallyknown in the art. In particular, purified LME may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind LME. Antibodies to LME may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are generally preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith LME or with any fragment or oligopeptide hereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to LME have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein. Short stretches of LME amino acids maybe fused with those of another protein, such as KLH, and antibodies tothe chimeric molecule may be produced.

Monoclonal antibodies to LME may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Sees e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S. P. etal. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce LME-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries.(See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for LME may alsobe generated. For example, such fragments include, but are not limitedto, F(ab′)₂ fragments produced by pepsin digestion of the antibodymolecule and Fab fragments generated by reducing the disulfide bridgesof the F(ab′)2 fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. (See, e.g., Huse, W. D. et al.(1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between LME and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering LME epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for LME. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of LME-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple LME epitopes, represents the average affinity,or avidity, of the antibodies for LME. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular LME epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theLME-antibody complex must withstand rigorous manipulations. Low-affinityantibody preparations with K_(a) ranging from about 10⁶ to 10⁷ L/moleare preferred for use in immunopurification and similar procedures whichultimately require dissociation of LME, preferably in active form, fromthe antibody (Catty, D. (1988) Antibodies, Volume I: A PracticalApproach, IRL Press, Washington D.C.; Liddell, J. E. and A. Cryer (1991)A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New YorkN.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/n, is generally employed in proceduresrequiring precipitation of LME-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingLME, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, modifications of gene expression can beachieved by designing complementary sequences or antisense molecules(DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding LME. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding LME. (See, e.g., Agrawal, S., ed. (1996) AntisenseTherapeutics, Humana Press Inc., Totawa N.J.)

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein. (See, e.g., Slater, J. E. et al. (1998) J. AllergyCli. Immunol. 102(3):469-475; and Scanlon, K. J. et al. (1995)9(13):1288-1296.) Antisense sequences can also be introducedintracellularly through the use of viral vectors, such as retrovirus andadeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.Ther. 63(3):323-347.) Other gene delivery mechanisms includeliposome-derived systems, artificial viral envelopes, and other systemsknown in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull.51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res.25(14):2730-2736.)

In another embodiment of the invention, polynucleotides encoding LME maybe used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesterolemia, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Soma (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HIV)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus(HBV, HCV); fungal parasites, such as Candida albicans andParacoccidioides brasiliensis; and protozoan parasites such asPlasmodium falciparum and Trypanosoma cruzi). In the case where agenetic deficiency in LME expression or regulation causes disease theexpression of LME from an appropriate population of transduced cells mayalleviate the clinical manifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in LME are treated by constructing mammalian expressionvectors encoding LME and introducing these vectors by mechanical meansinto LME-deficient cells. Mechanical transfer technologies for use withcells in vivo or ex vitro include (i) direct DNA microinjection intoindividual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;Boulay, J-L. and H. Récipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of LMEinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT, PCMV-TAG,PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF, PTET-ON, PTRE2,PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). LME may be expressedusing (i) a constitutively active promoter, (e.g., from cytomegalovirus(CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), orβ-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen); the ecdysone-inducible promoter (available in the plasmidsPVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; orthe RU486/mifepristone inducible promoter (Rossi, F. M. V. and Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native promoterof the endogenous gene encoding LME from a normal individual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to LME expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding LME under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci, USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 toRigg (“Method for obtaining retrovirus packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; SU, L. (1997)Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding LME to cells which have one ormore genetic abnormalities with respect to the expression of LME. Theconstruction and packaging of adenovirus-based vectors are well known tothose with ordinary skill in the art. Replication defective adenovirusvectors have proven to be versatile for importing genes encodingimmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially usefuladenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano(“Adenovirus vectors for gene therapy”), hereby incorporated byreference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999) Annu. Rev. Nutr. 19:511-544 and Verma, I. M. and N. Somia (1997)Nature 18:389:239-242, both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding LME to target cells which haveone or more genetic abnormalities with respect to the expression of LME.The use of herpes simplex virus (HSV)-based vectors may be especiallyvaluable for introducing LME to cells of the central nervous system, forwhich HSV has a tropism. The construction and packaging of herpes-basedvectors are well blown to those with ordinary skill in the art. Areplication-competent herpes simplex virus (HSV) type 1-based vector hasbeen used to deliver a reporter gene to the eyes of primates (Liu, X. etal. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virusvector has also been disclosed in detail in U.S. Pat. No. 5,804,413 toDeLuca (“Herpes simplex virus strains for gene transfer”), which ishereby incorporated by reference. U.S. Pat. No. 5,804,413 teaches theuse of recombinant HSV d92 which consists of a genome containing atleast one exogenous gene to be transferred to a cell under the controlof the appropriate promoter for purposes including human gene therapy.Also taught by this patent are the construction and use of recombinantHSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see alsoGoins, W. F. et alt (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994)Dev. Biol. 163:152-161, hereby incorporated by reference. Themanipulation of cloned herpesvirus sequences, the generation ofrecombinant virus following the transfection of multiple plasmidscontaining different segments of the large herpesvirus genomes, thegrowth and propagation of herpesvirus, and the infection of cells withherpesvirus are techniques well known to those of ordinary skill in theart.

In another alternative, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding LME to targetcells. The biology of the prototypic alphavirus, Semliki Forest Virus(SFV), has been studied extensively and gene transfer vectors have beenbased on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genomicRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for LME into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of LME-coding RNAs and the synthesis of high levels ofLME in vector transduced cells. While alphavirus infection is typicallyassociated with cell lysis within a few days, the ability to establish apersistent infection in hamster normal kidney cells (BHK-21) with avariant of Sindbis virus (SIN) indicates that the lytic replication ofalphaviruses can be altered to suit the needs of the gene therapyapplication (Dryga, S. A. et al. (1997) Virology 228:74-83). The widehost range of alphaviruses will allow the introduction of LME into avariety of cell types. The specific transduction of a subset of cells ina population may require the sorting of cells prior to transduction. Themethods of manipulating infectious cDNA clones of alphaviruses,performing alphavirus cDNA and RNA transfections, and performingalphavirus infections, are well known to those with ordinary skill inthe art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpalling is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, a regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature. (See,e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecularand Immunologic Approaches, Futura Publishing. Mt. Kisco N.Y., pp.163-177.) A complementary sequence or antisense molecule may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingLME.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding LME. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limed to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding LME. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased LMEexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding LME may be therapeuticallyuseful and in the treatment of disorders associated with decreased LMEexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding LME may be therapeuticallyuseful.

At least one, and up to a plurality, of test compounds may be screenedfor effectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding LME isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding LME are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingLME. The amount of hybridization may be quantified, thus forming thebasis for a comparison son of the expression of the polynucleotide bothwith and without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified-oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex viva. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g. Goldman, C. K. et al. (1997) Nat.Biotechnol. 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of LME,antibodies to LME, and mimetics, agonists, antagonists, or inhibitors ofLME.

The compositions utilized in this invention may be administered by anynumber of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal,intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid adry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery has the advantage of administrationwithout needle injection, and obviates the need for potentially toxicpenetration enhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracelluar delivery of macromolecules comprising LME or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, LME or a fragment thereofmay be joined to a short cationic N-terminal portion from the HIV Tat-1protein. Fusion proteins thus generated have been found to transduceinto the cells of all tissues, including the brain, in a mouse modelsystem (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example LME or fragments thereof, antibodies of LME, andagonists, antagonists or inhibitors of LME, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage win be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind LME may beused for the diagnosis of disorders characterized by expression of LME,or in assays to monitor patients being treated with LME or agonists,antagonists, or inhibitors of LME. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for LME include methods which utilizethe antibody and a label to detect LME in human body fluids or in exactsof cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring LME, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of LME expression. Normal or standard values for LMEexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to LME under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of LME expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding LMEmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantify gene expression in biopsied tissues in which expression of LMEmay be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of LME, and tomonitor regulation of LME levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding LME or closely related molecules may be used to identifynucleic acid sequences which encode LME. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif and the stringency of the hybridization or amplification willdetermine whether the probe identifies only naturally occurringsequences encoding LME, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the LME encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:7-12 or fromgenomic sequences including promoters, enhancers, and introns of the LMEgene.

Means for producing specific hybridization probes for DNAs encoding LMEinclude the cloning of polynucleotide, sequences encoding LME or LMEderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding LME may be used for the diagnosis ofdisorders associated with expression of LME. Examples of such disordersinclude, but are not limited to, cancer, such as adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous systemincluding Down syndrome, cerebral palsy, neuroskeletal disorders,autonomic nervous system disorders, cranial nerve disorders, spinal corddiseases, muscular dystrophy and other neuromuscular disorders,peripheral nervous system disorders, dermatomyositis and polymyositis,inherited, metabolic, endocrine, and toxic myopathies, myastheniagravis, periodic paralysis, mental disorders including mood, anxiety,and schizophrenic disorders, seasonal affective disorder (SAD),akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, Tourette'sdisorder, progressive supranuclear palsy, corticobasal degeneration, andfamilial frontotemporal dementia; an autoimmune/inflammatory disordersuch as acquired immunodeficiency syndrome (AIDS), Addison's disease,adult respiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma agastrointestinal disorder such as dysphagia, peptic esophagitis,esophageal spasm, esophageal structure, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquiredimmunodeficiency syndrome (AIDS) enteropathy, jaundice, hepaticencephalopathy, hepatorenal syndrome, hepatic steatosis,hemochromatosis, Wilson's disease, alpha₁-antitrypsin deficiency, Reye'ssyndrome, primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, centrilobular necrosis, peliosis hepatis,hepatic vein thrombosis, veno-occlusive disease, preeclampsia,eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis ofpregnancy, and hepatic tumors including nodular hyperplasias, adenomas,and carcinomas; and a cardiovascular disorder such as arteriovenousfistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease,aneurysms, arterial dissections, varicose veins, thrombophlebitis andphlebothrombosis, vascular tumors, and complications of thrombolysis,balloon angioplasty, vascular replacement, and coronary artery bypassgraft surgery, congestive heart failure, ischemic heart disease, anginapectoris, myocardial infarction, hypertensive heart disease,degenerative valvular heart disease, calcific aortic valve stenosis,congenitally bicuspid aortic valve, mitral annular calcification, mitralvalve prolapse, rheumatic fever and rheumatic heart disease, infectiveendocarditis, nonbacterial thrombotic endocarditis, endocarditis ofsystemic lupus erythematosus, carcinoid heart disease, cardiomyopathy,myocarditis, pericarditis, neoplastic heart disease, congenital heartdisease, and complications of cardiac transplantation, congenital lunganomalies, atelectasis, pulmonary congestion and edema, pulmonaryembolism, pulmonary hemorrhage, pulmonary infarction, pulmonaryhypertension, vascular sclerosis, obstructive pulmonary disease,restrictive pulmonary disease, chronic obstructive pulmonary disease,emphysema, chronic bronchitis, bronchial asthma, bronchiectasis,bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess,pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophiliabronchiolitis obliterans-organizing pneumonia, diffuse pulmonaryhemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonaryhemosiderosis, pulmonary involvement in collagen-vascular disorders,pulmonary alveolar proteinosis, lung tumors, inflammatory andnoninflammatory pleural effusions, pneumothorax, pleural tumors,drug-induced lung disease, radiation-induced lung disease, andcomplications of lung transplantation. The polynucleotide sequencesencoding LME may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; in dipstick,pin, and multiformat ELISA-like assays; and in microarrays utilizingfluids or tissues from patients to detect altered LME expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding LME may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingLME may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantified and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding LAW in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of LME, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding LME, under conditions suitablefor hybridization or amplification. Standard hybridization may bequantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding LME may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding LME, or a fragment of a polynucleotide complementary to thepolynucleotide encoding LME, and will be employed under optimizedconditions for identification of a specific gem or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived from thepolynucleotide sequences encoding LME may be used to detect singlenucleotide polymorphisms (SNPs). SNPs are substitutions, insertions anddeletions that are a frequent cause of inherited or acquired geneticdisease in humans. Methods of SNP detection include, but are not limitedto, single-stranded conformation polymorphism (SSCP) and fluorescentSSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from thepolynucleotide sequences encoding LME are used to amplify DNA using thepolymerase chain reaction (PCR). The DNA may be derived, for example,from diseased or normal tissue, biopsy samples, bodily fluids, and thelike. SNPs in the DNA cause differences in the secondary and tertiarystructures of PCR products in single-stranded form and these differencesare detectable using gel electrophoresis in non-denaturing gels. InfSCCP, the oligonucleotide primers are fluorescently labeled, whichallows detection of the amplimers in high-throughput equipment such asDNA sequencing machines. Additionally, sequence database analysismethods, termed in silico SNP (is SNP), are capable of identifyingpolymorphisms by comparing the sequence of individual overlapping DNAfragments which assemble into a common consensus sequence. Thesecomputer-based methods filter out sequence variations due to laboratorypreparation of DNA and sequencing errors using statistical models andautomated analyses of DNA sequence chromatograms. In the alternative,SNPs may be detected and characterized by mass spectrometry using, forexample, the high throughput MASSARRAY system (Sequenom, Inc., San DiegoCalif.).

Methods which may also be used to quantify the expression of LME includeradiolabeling or biotinylating nucleotides, coamplification of a controlnucleic acid, and interpolating results from standard curves. (See,e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin a high-throughput format where the oligomer or polynucleotide ofinterest is presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used aselements on a microarray. The microarray can be used in transcriptimaging techniques which monitor the relative expression levels of largenumbers of genes simultaneously as described below. The microarray mayalso be used to identify genetic variants, mutations, and polymorphisms.This information may be used to determine gene function, to understandthe genetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, LME, fragments of LME, or antibodies specific forLME may be used as elements on a microarray. The microarray may be usedto monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype A transcript image represents the global pattern of gene expressionby a particular tissue or cell type. Global gene expression patterns areanalyzed by quantifying the number of expressed genes and their relativeabundance under given conditions and at a given time. (See Seilhamer etal., “Comparative Gene Transcript Analysis,” U.S. Pat. No. 5,840,484,expressly incorporated by reference herein.) Thus a transcript image maybe generated by hybridizing the polynucleotides of the present inventionor their complements to the totality of transcripts or reversetranscripts of a particular tissue or cell type. In one embodiment, thehybridization takes place in high-throughput format, wherein thepolynucleotides of the present invention or their complements comprise asubset of a plurality of elements on a microarray. The resultanttranscript image would provide a profile of gene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471,expressly incorporated by reference herein). If a test compound has asignature similar to that of a compound with known toxicity, it islikely to share those toxic properties. These fingerprints or signaturesare most useful and refined when they contain expression informationfrom a large number of genes and gene families. Ideally, a genome-widemeasurement of expression provides the highest quality signature. Evengenes whose expression is not altered by any tested compounds areimportant as well, as the levels of expression of these genes are usedto normalize the rest of the expression data: The normalizationprocedure is useful for comparison of expression data after treatmentwith different compounds. While the assignment of gene function toelements of a toxicant signature aids in interpretation of toxicitymechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction oftoxicity. (See, for example, Press Release 00-02 from the NationalInstitute of Environmental Health Sciences, released Feb. 29, 2000,available from the internet.) Therefore, it is important and desirablein toxicological screening using toxicant signatures to include allexpressed gene sequences.

In one embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another particular embodiment relates to the use of the polypeptidesequences of the present invention to analyze the proteome of a tissueor cell type. The term proteome refers to the global pattern of proteinexpression in a particular tissue or cell type. Each protein componentof a proteome can be subjected individually to further analysis.Proteome expression patterns, or profiles, are analyzed by quantifyingthe number of expressed proteins and their relative abundance undergiven conditions and at a given time. A profile of a cell's proteome maythus be generated by separating and analyzing the polypeptides of aparticular tissue or cell type. In one embodiment, the separation isachieved using two-dimensional gel electrophoresis, in which proteinsfrom a sample are separated by isoelectric focusing in the firstdimension, and then according to molecular weight by sodium dodecylsulfate slab gel electophoresis in the second dimension (Steiner andAnderson, supra). The proteins are visualized in the gel as discrete anduniquely positioned spots, typically by staining the gel with an agentsuch as Coomassie Blue or silver or fluorescent stains. The opticaldensity of each protein spot is generally proportional to the level ofthe protein in the sample. The optical densities of equivalentlypositioned protein spots from different samples, for example, frombiological samples either treated or untreated with a test compound ortherapeutic agent, are compared to identify any changes in protein spotdensity related to the treatment. The proteins in the spots arepartially sequenced using, for example, standard methods employingchemical or enzymatic cleavage followed by mass spectrometry. Theidentity of the protein in a spot may be determined by comparing itspartial sequence, preferably of at least 5 contiguous amino acidresidues, to the polypeptide sequences of the present invention. In somecases, further sequence data may be obtained for definitive proteinidentification.

A proteomic profile may also be generated using antibodies specific forLME to quantify the levels of LME expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection may be performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inft art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Various types ofmicroarrays are well known and thoroughly described in DNA Microarrays:A Practical Approach, M. Schena, ed. (1999) Oxford University Press,London, hereby expressly incorporated by reference.

In another embodiment of the invention, nucleic acid sequences encodingLME may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. Either coding or noncodingsequences may be used, and in some, instances, noncoding sequences maybe preferable over coding sequences. For example, conservation of acoding sequence among members of a multi-gene family may potentiallycause undesired cross hybridization during chromosomal mapping. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries. (See, e.g., Harrington, J. J. et al.(1997) Nat. Genet 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.) Once mapped, thenucleic acid sequences of the invention may be used to develop geneticlinkage maps, for example, which correlate the inheritance of a diseasestate with the inheritance of a particular chromosome region orrestriction fragment length polymorphism (RFLP). (See, for example,Lander, E. S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA83:7353-7357.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995)in Meyers, supra, pp. 965-968.) Examples of genetic map data can befound in various scientific journals or at the Online MendelianInheritance in Man (OMIM) World Wide Web site. Correlation between thelocation of the gem encoding LME on a physical map and a specificdisorder, or a predisposition to a specific disorder, may help definethe region of DNA associated with that disorder and thus may furtherpositional cloning efforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation, (See, e.g., Gatti, R. A et alt (1988) Nature336:577-580.) The nucleotide sequence of the instant invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversions etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, LME, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between LMEand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geyser et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with LME, or fragments thereof and washed. Bound LME is thendetected by methods well known in the art. Purified LME can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding LME specificallycompete with a test compound for binding LME. In this manner, antibodiescan be used to detect the presence of any peptide which shares one ormore antigenic determinants with LME.

In additional embodiments, the nucleotide sequences which encode LME maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications and publications, mentionedabove and below, including U.S. Ser. No. 60/186,480, U.S. Ser. No.60/190,415, and U.S. Ser. No. 60/198,437, are expressly incorporated byreference herein.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs were dived from cDNA libraries described in the LIFESEQGOLD database (Incyte Genomics, Palo Alto Calif.) and shown in Table 4,column 5. Some tissues were homogenized and lysed in guanidiniumisothiocyanate, while others were homogenized and lysed in phenol or ina suitable mixture of denaturants, such as TRIZOL (Life Technologies), amonophasic solution of phenol and guanidine isothiocyanate. Theresulting lysates were centrifuged over CsCl cushions or extracted withchloroform. RNA was precipitated from the lysates with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+ RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRIPT plasmid system (Life Technologies), using the recommendedprocedures or similar methods known in the art. (See e.g., Ansubel,1997, supra, units 5.1-6.6.) Reverse transcription was initiated usingoligo d(T) or random primers. Synthetic oligonucleotide adapters wereligated to double stranded cDNA, and the cDNA was digested with theappropriate restriction enzyme or enzymes. For most libraries, the cDNAwas size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) orpreparative agarose gel electophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (LifeTechnologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), PBK-CMVplasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto Calif.), orderivatives thereof. Recombinant plasmids were transformed intocompetent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR fromStratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamPharmacia Biotech or supplied in ABI sequencing kits such as the ABIPRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (reviewed in Ausubel, 1997, supra,unit 7.7). Some of the cDNA sequences were selected for extension usingthe techniques disclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM,and hidden Markov model (HMM)-based protein family databases such asPFAM. (HMM is a probabilistic approach which analyzes consensus primarystructures of gene families. See, for example, Eddy, S. R. (1996) Curr.Opin. Struct. Biol. 6:361-365.) The queries were performed usingprograms based on BLAST, FASTA, BLIPS, and HMMER. The Incyte cDNAsequences were assembled to produce full length polynucleotidesequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitchedsequences, stretched sequences or Genscan-predicted coding sequences(see Examples IV and V) were used to extend Incyte cDNA assemblages tofull length. Assembly was performed using programs based on Phred,Phrap, and Consed, and cDNA assemblages were screened for open readingframes using programs based on GeneMark, BLAST, and PASTA. The fulllength polynucleotide sequences were translated to derive thecorresponding toll length polypeptide sequences. Alternatively, apolypeptide of the invention may begin at any of the methionine residuesof the full length translated polypeptide. Full length polypeptidesequences were subsequently analyzed by querying against databases suchas the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS,DOMO, PRODOM, Prosite, and hidden Markov model (HMM-based protein familydatabases such as PFAM. Full length polynucleotide sequences are alsoanalyzed using MACDNASIS PRO software (Hitachi Software Engineering,South San Francisco Calif.) and LASERGENE software (DNASTAR).Polynucleotide and polypeptide sequence alignments are generated usingdefault parameters specified by the CLUSTAL algorithm as incorporatedinto the MEGALIGN multisequence alignment program (DNASTAR), which alsocalculates the percent identity between aligned sequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysts and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:7-12.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 4.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative lipid metabolism enzymes were initially identified by run theGenscan gene identification program against public genomic sequencedatabases (e.g. gbpri and gbhtg). Genscan is a general-purpose geneidentification program which analyzes genomic DNA sequences from avariety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94, and Burge, C and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode lipid metabolism enzymes, the encoded polypeptides wereanalyzed by querying against PFAM models for lipid metabolism enzymes.Potential lipid metabolism enzymes were also identified by homology toIncyte cDNA sequences that had been annotated as lipid metabolismenzymes. These selected Genscan-predicted sequences were then comparedby BLAST analysis to the genpept and gbpri public databases. Thenecessary, the Genscan-predicted sequences were then edited bycomparison to the top BLAST hit from genpept to correct errors in thesequence predicted by Genscan, such as extra or omitted exons. BLASTanalysis was also used to find any Incyte cDNA or public cDNA coverageof the Genscan-predicted sequences, thus providing evidence fortranscription. When Incyte cDNA coverage was available, this informationwas used to correct or confirm the Genscan predicted sequence. Fulllength polynucleotide sequences were obtained by assemblingGenscan-predicted coding sequences with Incyte cDNA sequences and/orpublic cDNA sequences using the assembly process described in ExampleIII. Alternatively, full length polynucleotide sequences were derivedentirely from edited a unedited Genscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data

“Stitched” Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example III were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect exons predicted by Genscan wore corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pains (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegem.

VI. Chromosomal Mapping of LME Encoding Polynucleotides

The sequences which were used to assemble SEQ ID NO:7-12 were comparedwith sequences from the Incyte LIFESEQ database and public domaindatabases using BLAST and other implementations of the Smith-Watermanalgorithm. Sequences from these databases that matched SEQ ID NO:7-12were assembled into clusters of contiguous and overlapping sequencesusing assembly algorithms such as Phrap (Table 7). Radiation hybrid andgenetic mapping data available from public resources such as the StoredHuman Genome Center (SHGC), Whitehead Institute for Genome Research(WIGR), and Généthon were used to determine if any of the clusteredsequences had been previously mapped. Inclusion of a mapped sequence ina cluster resulted in the assignment of all sequences of that cluster,including its particular SEQ ID NO:, to that map location.

Map locations are represented by ranges, or intervals, or humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (ThecentiMorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Genethon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” on the internet, can be employed todetermine if previously identified disease genes map within or inproximity to the intervals indicated above.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel (1995) supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in cDNA databases such as GenBank orLIFESEQ (Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:

$\frac{{BLAST}\mspace{14mu}{Score} \times {Percent}\mspace{14mu}{Identity}}{5 \times {minimum}\left\{ {{{length}\mspace{11mu}\left( {{Seq}.\mspace{11mu} 1} \right)},{{length}\mspace{11mu}\left( {{Seq}.\mspace{11mu} 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,ten the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% to identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotide sequences encoding LME are analyzed withrespect to the tissue sources from which they were derived. For example,some full length sequences are assembled, at least in part, withoverlapping Incyte cDNA sequences (see Example III). Each cDNA sequenceis derived from a cDNA library constructed from a human tissue. Eachhuman tissue is classified into one of the following organ/tissuecategories: cardiovascular system; connective tissue; digestive system;embryonic structures; endocrine system exocrine glands; genitalia,female; genitalia, male; germ cells; hemic and immune system, liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding LME. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VIII. Extension of LME Encoding Polynucleotides

Full length polynucleotide sequences were also produced by extension ofan appropriate fragment of the full length molecule usingoligonucleotide primers designed from this fragment. One primer wassynthesized to initiate 5 extension of the known fragment, and the otherprimer was synthesized to initiate 3′ extension of the known fragment.The initial primers were designed using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 4° C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1×TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amersham Pharmacia Biotech). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase(Stratagene) to fill-in restriction site overhangs, and transfected intocompetent E. coli cells. Transformed cells were selected onantibiotic-contain media, and individual colonies were picked andcultured overnight at 37° C. in 384-well plates in LB/2× carb liquidmedia.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethylsulfoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Applied Biosystems).

In like manner, fall length polynucleotide sequences are verified usingthe above procedure or are used to obtain 5′ regulatory sequences usingthe above procedure along with oligonucleotides designed for suchextension, and an appropriate genomic library.

IX. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:7-12 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinaseDuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up tot for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

X. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting, See e.g., Baldeschweiler, supra.), mechanical microspottingtechnologies, and derivatives thereof. The substrate in each of theaforementioned technologies should be uniform and solid with anon-porous surface (Schena (1999), supra). Suggested substrates includesilicon, silica, glass slides, glass chips, and silicon wafers.Alternatively, a procedure analogous to a dot or slot blot may also beused to arrange and link elements to the surface of a substrate usingthermal, UV, chemical, or mechanical bonding procedures. A typical arraymay be produced using available methods and machines well known to thoseof ordinary skill in the art and may contain any appropriate number ofelements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A and J.Hodgson (1998) Nat. Biotechnol. 16:27-31.)

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorption and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (AmershamPharmacia Biotech). The reverse transcription reaction is performed in a25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits(Incyte). Specific control poly(A)⁺ RNAs are synthesized by in vitrotranscription from non-coding yeast genomic DNA. After incubation at 37°C. for 2 hr, each reaction sample (one with Cy3 and another with Cy5labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubatedfor 20 minutes at 85° C. to the stop the reaction and degrade the RNA.Samples are purified using two successive CHROMA SPIN 30 gel filtrationspin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto Calif.)and after combining, both reaction samples are ethanol precipitatedusing 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of100% ethanol. The sample is then dried to completion using a SpeedVAC(Savant Instruments Inc. Holbrook N.Y.) and resuspended in 14 μl5×SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sits areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 450 in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal win each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte).

XI. Complementary Polynucleotides

Sequences complementary to the LME-encoding sequences, or any partsthereof are used to detect, decrease, or inhibit expression of naturallyoccurring LME. Although use of oligonucleotides comprising from about 15to 30 base pairs is described, essentially the same procedure is usedwith smaller or with larger sequence fragments. Appropriateoligonucleotides are designed using OLIGO 4.06 software (NationalBiosciences) and the coding sequence of LME. To inhibit transcription, acomplementary oligonucleotide is designed from the most unique 5′sequence and used to prevent promoter binding to the coding sequence. Toinhibit translation, a complementary oligonucleotide is designed toprevent ribosomal binding to the LME-encoding transcript.

XII. Expression of LME

Expression and purification of LME is achieved using bacterial orvirus-based expression systems. For expression of LME in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express LME uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof LME in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, LME is synthesized as a fusion protein with,e.g., glutathione S-transferase (GST) or a peptide epitope tag, such asFLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from LME at specifically engineered sites. FLAG,an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch. 10 and 16). Purified LME obtained by these methods can beused directly in the assays shown in Examples XVI and XVII, whereapplicable.

XIII. Functional Assays

LME function is assessed by expressing the sequences encoding LME atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives nigh levels of cDNA expression. Vectors of choiceinclude PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,for example, an endothelial or hematopoietic cell line, using eitherliposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of +fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

The influence of LME on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding LMEand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding LME and other genes of interest canbe analyzed by northern analysis or microarray techniques.

XIV. Production of LME Specific Antibodies

LME substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the LME amino add sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art (See, e.g., Ausubel,1995, supra, ch. 11.)

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chand coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See e.g., Ausubel, 1995, supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide and anti-LME activity by,for example, binding the peptide or LME to a substrate, blocking with 1%BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XV. Purification of Naturally Occurring LME Using Specific Antibodies

Naturally occurring or recombinant LME is substantially purified byimmunoaffinity chromatography using antibodies specific for LME. Animmunoaffinity column is constructed by covalently coupling anti-LMEantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing LME are passed over the immunoaffinity column and thecolumn is washed under conditions that allow the preferential absorbanceof LME (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/LME binding(e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope,such as urea or thiocyanate ion), and LME is collect.

XVI. Identification of Molecules which Interact with LME

LME, or biologically active fragments thereof are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton A. E. and W. M. Hunter (1973)Biochem. J. 133:529-539.) Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled LME, washed,and any wells with labeled LME complex are assayed. Data obtained usingdifferent concentrations of LME are used to calculate values for thenumber, affinity, and association of LME with the candidate molecules.

Alternatively, molecules interacting with LME are analyzed using theyeast two-hybrid system as described in Fields, S. and O. Song (1989)Nature 340:245-246, or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (Clontech).

LME may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XVII. Demonstration of LME Activity

LME activity can be demonstrated by an in vitro hydrolysis assay withvesicles containing 1-palmitoyl-2-[1-¹⁴C]oleoyl phosphatidylcholine(Sigma-Aldrich). LME triglyceride lipase activity and phospholipase A₂activity are demonstrated by analysis of the cleavage products isolatedfrom the hydrolysis reaction mixture.

Vesicles containing 1-palmitoyl-2-[1-¹⁴C]oleoyl phosphatidylcholine(Amersham Pharmacia Biotech.) are prepared by mixing 2.0 μCi of theradiolabeled phospholipid with 12.5 mg of unlabeled 1-palmitoyl-2-oleoylphosphatidylcholine and drying the mixture under N₂. 2.5 ml of 150 mMTris-HCl, pH 7.5, is added, and the mixture is sonicated andcentrifuged. The supernatant may be stored at 4° C. The final reactionmixture is contain 0.25 ml of Hanks buffered salt solution supplementedwith 2.0 mM taurchenodeoxycholate, 1.0% bovine serum albumin, 1.0 mMCaCl₂, pH 7.4, 150 μg of 1-palmitoyl-2-[1-¹⁴C]oleoyl phosphatidylcholinevesicles, and various amounts of LME diluted in PBS. After incubationfor 30 min at 37° C., 20 μg each of lyso-phosphatidylcholine and oleicacid are added as carriers and each sample is extracted for totallipids. The lipids are separated by thin layer chromatography using atwo solvent system of choroform:methanol:acetic acid:water (65:35:8:4)until the solvent front is halfway up the plate. The process is thencontinued with hexane:ether:acetic acid (86:16:1) until the solventfront is at the top of the plate. The lipid-containing areas arevisualized with I₂ vapor; the spots are scraped, and their radioactivityis determined by scintillation counting. The amount of radioactivityreleased as fatty acids will increase as a greater amount of LME isadded to the assay mixture while the amount of radioactivity released aslyso-phosphatidylcholine will remain low. This demonstrates that LMEcleaves at the sn-2 and not the sn-1 position, as is characteristic ofphospholipase A₂ activity.

Alternatively, LME phospholipase activity is measured by the hydrolysisof a fatty acyl residue at the sn-1 position of phosphatidylserine. LMEis combined with the Tritium [³H] labeled substrate phosphatidylserineat stoichiometric quantities in a suitable buffer. Following anappropriate incubation time, the hydrolyzed reaction products areseparated from the substrates by chromatographic methods. The amount ofacylglycerophosphoserine produced is measured by counting tritiatedproduct with the help of a scintillation counter. Various control groupsare set up to account for background noise and unincorporated substrate.The final counts represent the tritiated enzyme product[³H]-acylglycerophosphoserine, which is directly proportional to theactivity of LME in biological samples.

LME lipoxygenase activity can be measured by chromatographic methods.Extracted LME lipoxygenase protein is incubated with 100 μM [1-¹⁴C]arachidonic acid or other unlabeled fatty acids at 37° C. for 30 min.After the incubation, stop solution (acetonitrile:methanol:water,350:150:1) is added. The samples are extracted and analyzed byreverse-phase HPLC by using a solvent system of methanol/water/aceticacid, 85:15:0.01 (vol/vol) at a flow rate of 1 ml/min. The effluent ismonitored at 235 nm and analyzed for the presence of the majorarachidonic metabolite such as 12-HPETE (catalyzed by 12-LOX). Thefractions are also subjected to liquid scintillation counting. The finalcounts represent the products, which is directly proportional to deactivity of LME in biological samples. For stereochemical analysis, themetabolites of arachidonic acid are analyzed her by chiral phase-HPLCand by mass spectrometry (Sun, D. et al. (1998) J. Biol. Chem.273:33540-33547).

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with certain embodiments,it should be understood that the invention as claimed should not beunduly limited to such specific embodiments. Indeed, variousmodifications of the described modes for carrying out the inventionwhich are obvious to those skilled in molecular biology or relatedfields are intended to be within the scope of the following claims.

TABLE 1 Incyte Incyte Incyte Polypeptide Polypeptide PolynucleotidePolynucleotide Project ID SEQ ID NO: ID SEQ ID NO: ID 2372651 12372651CD1 7 2372651CB1 2470792 2 2470792CD1 8 2470792CB1 1506182 31506182CD1 9 1506182CB1 2690842 4 2690842CD1 10 2690842CB1 5027764 55027764CD1 11 5027764CB1 2488174 6 2488174CD1 12 2488174CB1

TABLE 2 Incyte Polypeptide Polypeptide GenBank Probability GenBank SEQID NO: ID ID NO: Score Homolog 1 2372651CD1 g3876901 2.50E−56 Similarityto Human enoyl-CoA hydratase (SW: ECHM_HUMAN) [Caenorhabditis elegans]((1998) Science 282: 2012-2018) 2 2470792CD1 g7024433 0 Male sterilityprotein 2-like protein [Torpedo marmorata] 3 1506182CD1 g11245478 1.00E−162 Nicotinamide mononucleotide adenylyl transferase [Homosapiens] 4 2690842CD1 g6503307 3.20E−18 Phospholipid biosyntheticacyltransferase family member [Arabidopsis thaliana] (Neuwald, A. F.(1997) Curr. Biol. 7: R465-466) 5 5027764CD1 g4090960 1.40E−64Phosphatidylserine-specific phospholipase A1 [Homo sapiens] (Nagai, Y.et al. (1999) J. Biol. Chem. 274: 1153-11059) 6 2488174CD1 g7274380 1.00E−151 Group III secreted phospholipase A2 [Homo sapiens] (Valentin,E. et al. (2000) J. Biol. Chem. 275: 7492-7496)

TABLE 3 SEQ Incyte Amino Potential Potential Analytical ID PolypeptideAcid Phosphorylation Glycosylation Signature Sequences, Methods and NO:ID Residues Sites Sites Domains and Motifs Databases 1 2372651CD1 303S49 S167 T233 Enoyl-CoA hydratase/isomerase ProfileScan S43 T69 S105signature: S210 F125-L181 Enoyl-CoA hydratase/isomerase HMMER-PFAMfamily: R57-P225 Enoyl-CoA hydratase/isomerase: BLIMPS- BL00166A:G55-K66 BLOCKS BL00166B: V93-E114 BL00166C: V140-A166 BL00166D:L190-P225 BL00166E: A289-R294 Enoyl CoA hydratase isomerase: BLAST-PD000432: I59-P225 PRODOM Enoyl-CoA hydratase/isomerase: BLAST-DOMODM00366|P30084|34-285: I59-K295 2 2470792CD1 515 T33 S94 T235 N128 N341ATP/GTP-binding site motif A (P- MOTIFS S389 S414 T114 N396 loop): T120T193 T257 A5-K11 S356 T371 T512 Male sterility protein 2: BLAST- Y168Y404 PD018334: W200-L445 PRODOM 3 1506182CD1 279 T130 S109 S256 N36Signal cleavage: M1-N22 SPScan S4 T38 T95 S136 Lipopolysaccharide coreBLIMPS- S223 S235 biosynthesis protein signature: PRINTS PR01020A:V9-L27 PR01020C: H65-Q89 Protein F26H9.4: BLAST- PD023338: V9-L106PRODOM Membrane protein YLR328W: BLAST-DOMO DM07979|P53204|1-394:M1-L106 4 2690842CD1 432 S35 S74 S76 T84 N111 Phospholipid biosyntheticHMMER-PFAM S176 S178 S203 acyltransferase: T208 S287 S333 R18-S203 T353T370 T394 S398 S80 T110 T117 S254 Y253 5 5027764CD1 451 T72 S14 S16 S97N50 N58 N66 Vespid venom allergen PLA1 BLIMPS- S144 T175 S206 N357signature PR00825A: PRINTS S258 S272 S287 P188-H205, L211-P231 T320 T47T68 Lipase precursor, signal, BLAST- S121 T124 T348 hydrolase, lipiddegradation, PRODOM glycoprotein PD001492: N64-I335 Triacylglycerollipase: BLAST-DOMO DM00344|A49488|25-326: L39-V333 Signal peptide:M1-A18 HMMER Signal cleavage: M1-A18 SPScan Lipase: H31-D319 HMMER-PFAMLipases, serine proteins BL00120: BLIMPS- N102-I116, D146-S160,Y223-C233 BLOCKS Triacylglycerol lipase family BLIMPS- signaturePR00821: PRINTS S59-F78, V104-H119, D147-E165, C246-E261, P325-K340 62488174CD1 312 S125 S57 S190 N15 N83 N128 Phospholipase A2 histidineactive MOTIFS S212 S87 S96 N199 N242 site (PDOC00109): S121 S138 S166C28-C35 T201 S253 Phospholipase A2 isozymes: BLAST- PD033132: M1-R66PRODOM A2, phospholipase, histidine: BLAST-DOMO DM05541|P80003|1-141:M1-R66 DM05541|B56338|1-136: P2-R66

TABLE 4 Incyte Polynucleotide Polynucleotide Sequence Selected Sequence5′ 3′ SEQ ID NO: ID Length Fragments Fragments Position Position 72372651CB1 1667 1-177 6827519J1 (SINTNOR01) 235 827 7001351H1(HEALDIR01) 531 1136 5601906H1 (UTRENON03) 1339 1652 6515361H1(THYMDIT01) 1116 1650 5602106H1 (UTRENON03) 1340 1653 3574443H1(BRONNOT01) 1 297 8 2470792CB1 2124 1-863, 70774783V1 1678 20662075-2124 70780150V1 682 1195 70067901V1 1548 2065 70067625V1 1209 16856830177J1 (SINTNOR01) 1 688 70781158V1 633 1157 70778451V1 946 1600622417H1 (PGANNOT01) 1871 2124 9 1506182CB1 2955 1-52, 1433938R1(BEPINON01) 1718 2183 550-587, 70047889V1 1439 1983 2567-2955, 1257710F6(MENITUT03) 2437 2955 1065-2018 6884818J1 (BRAHTDR03) 1947 26053284318F6 (HEAONOT05) 10 617 662677R6 (BRAINOT03) 275 841 1308778H1(COLNFET02) 1 261 6969331U1 742 1505 662677T6 (BRAINOT03) 1050 1640 102690842CB1 1579 1-313 2690842H1 (LUNGNOT23) 969 1210 7362670H1(LUNLTUE02) 1 615 7261254H1 (UTRETMC01) 1010 1579 6913602H1 (PITUDIR01)587 1043 11 5027764CB1 3170 2450-2737, 841054T6 (PROSTUT05) 2557 3170507-1843 3224086R6 (COLNNON03) 2477 3021 70705531V1 1621 2291 4438670H1(SINTNOT22) 1 272 6933434H1 (SINTTMR02) 14 652 70635824V1 2273 289270750908V1 1723 2330 70743692V1 999 1595 71051604V1 470 1054 71246013V11078 1692 12 2488174CB1 1900 1-1772 70906023V1 1368 1889 71530085V1 4971187 71531355V1 566 1284 70905361V1 1263 1888 2488174F6 (LUNGNOT22) 1532 71527244V1 1426 1899

TABLE 5 Polynucleotide Incyte Representative SEQ ID NO: Project IDLibrary 7 2372651CB1 PROSNOT18 8 2470792CB1 EOSIHET02 9 1506182CB1BRAITUT02 10 2690842CB1 UTRETMC01 11 5027764CB1 PROSTUT05 12 2488174CB1LUNGNOT22

TABLE 6 Library Vector Library Description BRAITUT02 PSPORT1 Library wasconstructed using RNA isolated from brain tumor tissue removed from thefrontal lobe of a 58-year-old Caucasian male during excision of acerebral meningeal lesion. Pathology indicated a grade 2 metastatichypernephroma. Patient history included a grade 2 renal cell carcinoma,insomnia, and chronic airway obstruction. Family history included amalignant neoplasm of the kidney. EOSIHET02 PBLUESCRIPT Library wasconstructed using RNA isolated from peripheral blood cells apheresedfrom a 48-year-old Caucasian male. Patient history includedhypereosinophilia. The cell population was determined to be greater than77% eosinophils by Wright's staining. LUNGNOT22 pINCY Library wasconstructed using RNA isolated from lung tissue removed from a 58-year-old Caucasian female. The tissue sample used to construct this librarywas found to have tumor contaminant upon microscopic examination.Pathology for the associated tumor tissue indicated a caseatinggranuloma. Family history included congestive heart failure, breastcancer, secondary bone cancer, acute myocardial infarction andatherosclerotic coronary artery disease. PROSNOT18 pINCY Library wasconstructed using RNA isolated from diseased prostate tissue removedfrom a 58-year-old Caucasian male during a radical cystectomy, radicalprostatectomy, and gastrostomy. Pathology indicated adenofibromatoushyperplasia; this tissue was associated with a grade 3 transitional cellcarcinoma. Patient history included angina and emphysema. Family historyincluded acute myocardial infarction, atherosclerotic coronary arterydisease, and type II diabetes. PROSTUT05 PSPORT1 Library was constructedusing RNA isolated from prostate tumor tissue removed from a 69-year-oldCaucasian male during a radical prostatectomy. Pathology indicatedadenocarcinoma (Gleason grade 3 + 4). Adenofibromatous hyperplasia wasalso present. Family history included congestive heart failure, multiplemyeloma, hyperlipidemia, and rheumatoid arthritis. UTRETMC01 pINCY Thislarge size-fractionated library was constructed using pooled cDNA fromtwo different donors. cDNA was generated using mRNA isolated fromendometrial tissue removed from a 32-year-old Caucasian female (donor A)during total abdominal hysterectomy, bilateral salpingo-oophorectomy,and cystocele repair; and from endometrial tissue removed from a48-year-old Caucasian female (donor B) during a vaginal hysterectomy,rectocele repair, and bilateral salpingo-oophorectomy. Pathology fordonor A indicated the endometrium was in the proliferative phase. Theright ovary showed a corpus luteal cyst. For donor B, pathologyindicated chronic cervicitis and the endometrium was weaklyproliferative. The right ovary and specimen from the peritoneumindicated endometriosis focally involving the surface of the right ovaryand the peritoneum. Pathology for the matched tumor tissue indicated asingle submucosal leiomyoma, which exhibited extensive hyalin changewith hyalin-type necrosis. The left ovary contained a corpus luteumcyst. Donor A presented with abdominal pain, stress incontinence, anddysmenorrhea. Patient history included hemorrhagic ovarian cysts,uterine endometriosis, normal delivery, and cesarean deliveries. Donor Bpresented with metrorrhagia, extrinsic asthma, depressive disorder, andanxiety state. Patient history included alcohol abuse, hyperlipidemia, anormal delivery, tobacco abuse in remission, and meningitis. Patientmedications (B) included Prozac, Trazodone, Clorazepate. and Medrol.

TABLE 7 Program Description Reference Parameter Threshold ABI A programthat removes vector sequences and Applied Biosystems, Foster City, CA.FACTURA masks ambiguous bases in nucleic acid sequences. ABI/ A FastData Finder useful in comparing and Applied Biosystems, Foster City, CA;Mismatch <50% PARACEL annotating amino acid or nucleic acid sequences.Paracel Inc., Pasadena, CA. FDF ABI A program that assembles nucleicacid sequences. Applied Biosystems, Foster City, CA. AutoAssembler BLASTA Basic Local Alignment Search Tool useful in Altschul, S. F. et al.(1990) J. Mol. Biol. ESTs: Probability sequence similarity search foramino acid and 215: 403-410; Altschul, S. F. et al. (1997) value =1.0E−8 or less nucleic acid sequences. BLAST includes five Nucleic AcidsRes. 25: 3389-3402. Full Length functions: blastp, blastn, blastx,tblastn, and tblastx. sequences: Probability value = 1.0E−10 or lessFASTA A Pearson and Lipman algorithm that searches for Pearson, W. R.and D. J. Lipman (1988) Proc. ESTs: fasta E value = similarity between aquery sequence and a group of Natl. Acad Sci. USA 85: 2444-2448;Pearson, 1.06E−6 Assembled sequences of the same type. FASTA comprisesas W. R. (1990) Methods Enzymol. 183: 63-98; ESTs: fasta Identity =least five functions: fasta, tfasta, fastx, tfastx, and and Smith, T. F.and M. S. Waterman (1981) 95% or greater and Match ssearch. Adv. Appl.Math. 2: 482-489. length = 200 bases or greater; fastx E value = 1.0E−8or less Full Length sequences: fastx score = 100 or greater BLIMPS ABLocks IMProved Searcher that matches a Henikoff, S. and J. G. Henikoff(1991) Nucleic Probability value = sequence against those in BLOCKS,PRINTS, Acids Res. 19: 6565-6572; Henikoff, J. G. and 1.0E−3 or lessDOMO, PRODOM, and PFAM databases to search S. Henikoff (1996) MethodsEnzymol. for gene families, sequence homology, and structural 266:88-105; and Attwood, T. K. et al. (1997) J. fingerprint regions. Chem.Inf. Comput. Sci. 37: 417-424. HMMER An algorithm for searching a querysequence against Krogh, A. et al. (1994) J. Mol. Biol. PFAM hits:Probability hidden Markov model (HMM)-based databases of 235: 1501-1531;Sonnhammer, E. L. L. et al. value = 1.0E−3 or less protein familyconsensus sequences, such as PFAM. (1988) Nucleic Acids Res. 26:320-322; Signal peptide hits: Durbin, R. et al. (1998) Our World View,in a Score = 0 or greater Nutshell, Cambridge Univ. Press, pp. 1-350.ProfileScan An algorithm that searches for structural and sequenceGribskov, M. et al. (1988) CABIOS 4: 61-66; Normalized quality motifs inprotein sequences that match sequence Gribskov, M. et al. (1989) MethodsEnzymol. score ≧ GCG-specified patterns defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) “HIGH” value for Nucleic Acids Res.25: 217-221. that particular Prosite motif. Generally, score = 1.4-2.1.Phred A base-calling algorithm that examines automated Ewing, B. et al.(1998) Genome Res. sequencer traces with high sensitivity andprobability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194. Phrap A Phils Revised Assembly Program including SWAT Smith, T.F. and M. S. Waterman (1981) Adv. Score = 120 or and CrossMatch,programs based on efficient Appl. Math. 2: 482-489; Smith, T. F. and M.S. greater; Match length = implementation of the Smith-Watermanalgorithm, Waterman (1981) J. Mol. Biol. 147: 195-197; 56 or greateruseful in searching sequence homology and and Green, P., University ofWashington, assembling DNA sequences. Seattle, WA. Consed A graphicaltool for viewing and editing Phrap Gordon, D. et al. (1998) Genome Res.8: assemblies. 195-202. SPScan A weight matrix analysis program thatscans protein Nielson, H. et al. (1997) Protein Engineering Score = 3.5or greater sequences for the presence of secretory signal peptides. 10:1-6; Claverie, J. M. and S. Audic (1997) CABIOS 12: 431-439. TMAP Aprogram that uses weight matrices to delineate Persson, B. and P. Argos(1994) J. Mol. Biol. transmembrane segments on protein sequences and237: 182-192; Persson, B. and P. Argos (1996) determine orientation.Protein Sci. 5: 363-371. TMHMMER A program that uses a hidden Markovmodel (HMM) Sonnhammer, E. L. et al. (1998) Proc. Sixth Intl. todelineate transmembrane segments on protein Conf. on Intelligent Systemsfor Mol. Biol., sequences and determine orientation. Glasgow et al.,eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA,pp. 175-182. Motifs A program that searches amino acid sequences forBairoch, A. et al. (1997) Nucleic Acids Res. 25: patterns that matchedthose defined in Prosite. 217-221; Wisconsin Package Program Manual,version 9, page M51-59, Genetics Computer Group, Madison, WI.

1. An isolated polynucleotide encoding a polypeptide comprising theamino acid of SEQ ID NO: 5, wherein: a) the polynucleotide comprises thesequence of SEQ ID NO: 11; and b) the polypeptide hasphosphatidylserine-specific phospholipase A 1 activity.
 2. A recombinantpolynucleotide comprising a promoter sequence operably linked to thepolynucleotide of claim
 1. 3. An isolated cell transformed with therecombinant polynucleotide of claim
 2. 4. A method of producing anisolated polypeptide that is encoded by the polynucleotide of claim 1and that has phosphotidylserine-specific phospholipase A1 activity, themethod comprising: a) culturing a cell under conditions suitable forexpression of the polypeptide, wherein said cell is transformed with arecombinant polynucleotide, and said recombinant polynucleotide comprisea promoter sequence operably linked to the polynucleotide of claim 1,and b) recovering the polypeptide so expressed.
 5. An isolatedpolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of: a) a polynucleotide sequence having at least about95% sequence identity to the polynucleotide sequence of SEQ ID NO: 11,wherein the polynucleotide encodes a polypeptide havingphosphatidylserine-specific phospholipase A 1 activity; and b) apolynucleotide sequence complementary to the polynucleotide sequence ofa.